Anti-nr10 antibody and use thereof

ABSTRACT

The present inventors successfully obtained anti-NR10 antibodies having an effective neutralizing activity against NR10. The anti-NR10 antibodies provided by the present invention are useful as, for example, pharmaceuticals for treating or preventing inflammatory diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/340,883, filed Jul. 25, 2014, which is a continuation of U.S.application Ser. No. 12/745,781, filed Sep. 13, 2010 (now abandoned),which is a 371 of PCT/JP2008/072152, filed Dec. 5, 2008, which in turnclaims the benefit of Japanese Patent Application Nos. 2007-315143,filed Dec. 5, 2007, and 2008-247425, filed Sep. 26, 2008.

TECHNICAL FIELD

The present invention relates to anti-NR10 antibodies, andpharmaceutical compositions comprising an anti-NR10 antibody.

BACKGROUND ART

Many cytokines are known as humoral factors involved in the growth anddifferentiation of various types of cells, or in the activation ofdifferentiated mature cell functions. Cytokine-stimulated cells producedifferent types of cytokines, thereby forming networks of multiplecytokines in the body. Biological homeostasis is maintained by adelicate balance of the mutual regulation between cytokines in thesenetworks. Many inflammatory diseases are thought to result from afailure of such cytokine networks. Thus, monoclonal antibody-basedanti-cytokine therapy is drawing much attention. For example, anti-TNFantibodies and anti-IL-6 receptor antibodies have been demonstrated tobe highly effective clinically. On the other hand, there are manyexamples of failure where no therapeutic effects were produced when asingle cytokine, such as IL-4, was blocked alone, due to the activationof compensatory pathways in actual pathological conditions.

The present inventors succeeded in isolating a novel cytokine receptorNR10 that was highly homologous to gp130, a receptor for IL-6 signaltransduction (Patent Document 1). NR10 forms a heterodimer withoncostatin M receptor (OSMR) and functions as an IL-31 receptor(Non-patent Document 1). Regarding IL-31, it has been reported thattransgenic mice overexpressing IL-31 spontaneously develop pruriticdermatitis (Patent Document 2).

Antibodies that bind to NR10 and inhibit the binding between NR10 andIL-31 may be effective in treating inflammatory diseases. For clinicaluse, anti-NR10 antibodies are required to have low immunogenicity.Furthermore, in order to achieve high therapeutic effects, antibodieswith strong NR10-binding or neutralizing activity are desired.

Prior art documents of the present invention are described below.

-   Patent Document 1: WO00/75314-   Patent Document 2: WO03/060090-   Non-patent Document 1: IL-31 is associated with cutaneous lymphocyte    antigen-positive skin homing T cells in patients with atopic    dermatitis, J Allergy Clin Immunol. 2006 February; 117(2): 418-25.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the circumstancesdescribed above. An objective of the present invention is to provideanti-NR10 antibodies, and pharmaceutical compositions comprising ananti-NR10 antibody.

Means for Solving the Problems

The present inventors conducted dedicated studies to achieve theobjective described above. The present inventors succeeded in obtaininganti-NR10 antibodies having an effective neutralizing activity againstNR10. Furthermore, the present inventors succeeded in humanizing theantibodies while maintaining their activity. The present inventors alsosuccessfully produced antibodies with improved pharmacokinetics,enhanced antigen-binding activity, improved stability, and/or reducedrisk of immunogenicity. These antibodies are useful as therapeuticagents for inflammatory diseases.

The present invention relates to anti-NR10 antibodies, andpharmaceutical compositions comprising an anti-NR10 antibody. Morespecifically, the present invention includes:

[1] an antibody that recognizes domain 1 of NR10;[2] the antibody of [1], which has a neutralizing activity;[3] the antibody of [1] or [2], which is a humanized antibody;[4] an anti-NR10 antibody which is any one of:(1) an antibody comprising a heavy chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprisingthe amino acid sequence of SEQ ID NO: 2, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 3;(2) an antibody comprising the heavy chain variable region of SEQ ID NO:4;(3) an antibody comprising a light chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 5, CDR2 comprisingthe amino acid sequence of SEQ ID NO: 6, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 7;(4) an antibody comprising the light chain variable region of SEQ ID NO:8;(5) an antibody comprising the heavy chain variable region of (1) andthe light chain variable region of (3);(6) an antibody comprising the heavy chain variable region of (2) andthe light chain variable region of (4);(7) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (1) to(6), which has an activity equivalent to that of the antibody of any oneof (1) to (6); and(8) an antibody which binds to the same epitope as an epitope bound bythe antibody of any one of (1) to (7);[5] an anti-NR10 antibody which is any one of:(1) an antibody comprising a heavy chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprisingthe amino acid sequence of SEQ ID NO: 10, and CDR3 comprising the aminoacid sequence of SEQ ID NO: 11;(2) an antibody comprising the heavy chain variable region of SEQ ID NO:12;(3) an antibody comprising a light chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 13, CDR2comprising the amino acid sequence of SEQ ID NO: 14, and CDR3 comprisingthe amino acid sequence of SEQ ID NO: 15;(4) an antibody comprising the light chain variable region of SEQ ID NO:16;(5) an antibody comprising the heavy chain variable region of (1) andthe light chain variable region of (3);(6) an antibody comprising the heavy chain variable region of (2) andthe light chain variable region of (4);(7) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (1) to(6), which has an activity equivalent to that of the antibody of any oneof (1) to (6); and(8) an antibody which binds to the same epitope as an epitope bound bythe antibody of any one of (1) to (7);[6] an anti-NR10 antibody which is any one of:(1) an antibody comprising a heavy chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 17, CDR2comprising the amino acid sequence of SEQ ID NO: 18, and CDR3 comprisingthe amino acid sequence of SEQ ID NO: 19;(2) an antibody comprising the heavy chain variable region of SEQ ID NO:20;(3) an antibody comprising a light chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 21, CDR2comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprisingthe amino acid sequence of SEQ ID NO: 23;(4) an antibody comprising the light chain variable region of SEQ ID NO:24;(5) an antibody comprising the heavy chain variable region of (1) andthe light chain variable region of (3);(6) an antibody comprising the heavy chain variable region of (2) andthe light chain variable region of (4);(7) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (1) to(6), which has an activity equivalent to that of the antibody of any oneof (1) to (6); and(8) an antibody which binds to the same epitope as an epitope bound bythe antibody of any one of (1) to (7);[7] an anti-NR10 antibody which is any one of:(1) an antibody comprising a heavy chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 25, CDR2comprising the amino acid sequence of SEQ ID NO: 26, and CDR3 comprisingthe amino acid sequence of SEQ ID NO: 27;(2) an antibody comprising the heavy chain variable region of SEQ ID NO:28;(3) an antibody comprising a light chain variable region which comprisesCDR1 comprising the amino acid sequence of SEQ ID NO: 29, CDR2comprising the amino acid sequence of SEQ ID NO: 30, and CDR3 comprisingthe amino acid sequence of SEQ ID NO: 31;(4) an antibody comprising the light chain variable region of SEQ ID NO:32;(5) an antibody comprising the heavy chain variable region of (1) andthe light chain variable region of (3);(6) an antibody comprising the heavy chain variable region of (2) andthe light chain variable region of (4);(7) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (1) to(6), which has an activity equivalent to that of the antibody of any oneof (1) to (6); and(8) an antibody which binds to the same epitope as an epitope bound bythe antibody of any one of (1) to (7);[8] an antibody or antibody variable region which is any one of:(1) a heavy chain variable region comprising CDR1 of SEQ ID NO: 196,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 11 (H17);(2) a heavy chain variable region comprising CDR1 of SEQ ID NO: 176,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 11 (H19);(3) a heavy chain variable region comprising CDR1 of SEQ ID NO: 196,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 184 (H28, H42);(4) a heavy chain variable region comprising CDR1 of SEQ ID NO: 9, CDR2of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 184 (H30, H44);(5) a heavy chain variable region comprising CDR1 of SEQ ID NO: 176,CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 184 (H34, H46);(6) a heavy chain variable region comprising CDR1 of SEQ ID NO: 9, CDR2of SEQ ID NO: 198, and CDR3 of SEQ ID NO: 184 (H57, H78);(7) a heavy chain variable region comprising CDR1 of SEQ ID NO: 176,CDR2 of SEQ ID NO: 198, and CDR3 of SEQ ID NO: 184 (H71, H92);(8) a heavy chain variable region comprising CDR1 of SEQ ID NO: 9, CDR2of SEQ ID NO: 199, and CDR3 of SEQ ID NO: 184 (H97, H98);(9) a light chain variable region comprising CDR1 of SEQ ID NO: 200,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L11);(10) a light chain variable region comprising CDR1 of SEQ ID NO: 201,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L12);(11) a light chain variable region comprising CDR1 of SEQ ID NO: 202,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L17);(12) a light chain variable region comprising CDR1 of SEQ ID NO: 203,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L50);(13) an antibody comprising the heavy chain variable region of (3) andthe light chain variable region of (11);(14) an antibody comprising the heavy chain variable region of (4) andthe light chain variable region of (11);(15) an antibody comprising the heavy chain variable region of (5) andthe light chain variable region of (11);(16) an antibody comprising the heavy chain variable region of (6) andthe light chain variable region of (11);(17) an antibody comprising the heavy chain variable region of (7) andthe light chain variable region of (11);(18) an antibody comprising the heavy chain variable region of (8) andthe light chain variable region of (12);(19) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (13) to(18), which has an activity equivalent to that of the antibody of anyone of (13) to (18); and(20) an antibody which binds to the same epitope as an epitope bound bythe antibody of any one of (13) to (18);[9] an antibody or antibody variable region which is any one of: (1) aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 204 (H17);(2) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 205 (H19);(3) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 206 (H28);(4) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 207 (H30);(5) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 208 (H34),(6) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 209 (H42);(7) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 210 (H44);(8) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 211 (H46);(9) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 212 (H57);(10) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 213 (H71);(11) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 214 (H78);(12) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 215 (H92);(13) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 216 (H97);(14) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 217 (H98);(15) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 218 (L11);(16) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 219 (L12);(17) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 220 (L17);(18) a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 221 (L50);(19) an antibody comprising the heavy chain variable region of (3) andthe light chain variable region of (17) (H28L17);(20) an antibody comprising the heavy chain variable region of (4) andthe light chain variable region of (17) (H30L17);(21) an antibody comprising the heavy chain variable region of (5) andthe light chain variable region of (17) (H34L17);(22) an antibody comprising the heavy chain variable region of (6) andthe light chain variable region of (17) (H42L17);(23) an antibody comprising the heavy chain variable region of (7) andthe light chain variable region of (17) (H44L17);(24) an antibody comprising the heavy chain variable region of (8) andthe light chain variable region of (17) (H46L17);(25) an antibody comprising the heavy chain variable region of (9) andthe light chain variable region of (17) (H57L17);(26) an antibody comprising the heavy chain variable region of (10) andthe light chain variable region of (17) (H71L17);(27) an antibody comprising the heavy chain variable region of (11) andthe light chain variable region of (17) (H78L17);(28) an antibody comprising the heavy chain variable region of (12) andthe light chain variable region of (17) (H92L17);(29) an antibody comprising the heavy chain variable region of (13) andthe light chain variable region of (18) (H97L50);(30) an antibody comprising the heavy chain variable region of (14) andthe light chain variable region of (18) (H98L50),(31) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (19) to(30), which has an activity equivalent to that of the antibody of anyone of (19) to (30); and(32) an antibody which binds to the same epitope as an epitope bound bythe antibody of any one of (19) to (30);[10] the anti-NR10 antibody of any one of [4] to [9], which is ahumanized antibody;[11] an antibody, antibody heavy chain, or antibody light chain, whichis any one of:(1) a heavy chain comprising the amino acid sequence of SEQ ID NO: 222(H17);(2) a heavy chain comprising the amino acid sequence of SEQ ID NO: 223(H19);(3) a heavy chain comprising the amino acid sequence of SEQ ID NO: 224(H28);(4) a heavy chain comprising the amino acid sequence of SEQ ID NO: 225(H30);(5) a heavy chain comprising the amino acid sequence of SEQ ID NO: 226(H34);(6) a heavy chain comprising the amino acid sequence of SEQ ID NO: 227(H42);(7) a heavy chain comprising the amino acid sequence of SEQ ID NO: 228(H44);(8) a heavy chain comprising the amino acid sequence of SEQ ID NO: 229(H46);(9) a heavy chain comprising the amino acid sequence of SEQ ID NO: 230(H57);(10) a heavy chain comprising the amino acid sequence of SEQ ID NO: 231(H71);(11) a heavy chain comprising the amino acid sequence of SEQ ID NO: 232(H78);(12) a heavy chain comprising the amino acid sequence of SEQ ID NO: 233(H92);(13) a heavy chain comprising the amino acid sequence of SEQ ID NO: 234(H97);(14) a heavy chain comprising the amino acid sequence of SEQ ID NO: 235(H98);(15) a light chain comprising the amino acid sequence of SEQ ID NO: 236(L11);(16) a light chain comprising the amino acid sequence of SEQ ID NO: 237(L12);(17) a light chain comprising the amino acid sequence of SEQ ID NO: 238(L17);(18) a light chain comprising the amino acid sequence of SEQ ID NO: 239(L50);(19) an antibody comprising the heavy chain of (3) and the light chainof (17) (H28L17);(20) an antibody comprising the heavy chain of (4) and the light chainof (17) (H30L17);(21) an antibody comprising the heavy chain of (5) and the light chainof (17) (H34L17);(22) an antibody comprising the heavy chain of (6) and the light chainof (17) (H42L17);(23) an antibody comprising the heavy chain of (7) and the light chainof (17) (H44L17);(24) an antibody comprising the heavy chain of (8) and the light chainof (17) (H46L17);(25) an antibody comprising the heavy chain of (9) and the light chainof (17) (H57L17);(26) an antibody comprising the heavy chain of (10) and the light chainof (17) (H71L17);(27) an antibody comprising the heavy chain of (11) and the light chainof (17) (H78L17);(28) an antibody comprising the heavy chain of (12) and the light chainof (17) (H92L17);(29) an antibody comprising the heavy chain of (13) and the light chainof (18) (H97L50);(30) an antibody comprising the heavy chain of (14) and the light chainof (18) (H98L50);(31) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (19) to(30), which has an activity equivalent to that of the antibody of anyone of (19) to (30); and(32) an antibody which binds to the same epitope as an epitope bound bythe antibody of any one of (19) to (30);[12] a pharmaceutical composition comprising the antibody of any one of[1] to [11];[13] the pharmaceutical composition of [12], which is an agent fortreating an inflammatory disease;[14] a method for treating or preventing an inflammatory disease, whichcomprises the step of administering the antibody of any one of [1] to[11]; and[15] use of the antibody of any one of [1] to [11] in the preparation ofa therapeutic agent for an inflammatory disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of the heavy chain variableregions of mouse antibodies NS18, NS22, NS23, and NS33.

FIG. 2 shows the amino acid sequences of the light chain variableregions of mouse antibodies NS18, NS22, NS23, and NS33.

FIG. 3 is a graph showing the inhibition of hNR10/hOSMR/BaF3 cell growthby hybridoma culture supernatants.

FIG. 4 is a graph showing the inhibition of cynNR10/cynOSMR/BaF3 cellgrowth by hybridoma culture supernatants.

FIG. 5 is a graph showing the assessment of the activity of chimericNS22 (BaF).

FIG. 6 is a graph showing the assessment of the activity of chimericNS22 (DU-145).

FIG. 7 is a graph showing the assessment of the competition of chimericNS22 with IL-31.

FIG. 8 is a graph showing the NR10 competitive binding activity ofanti-NR10 antibodies.

FIG. 9 is a set of graphs showing the assessment of the competition ofhumanized NS22 (H0L0) with IL-31.

FIG. 10 shows the effect of the constant region of humanized anti-NR10antibody H0L0 on the heterogeneity assessed by cation exchangechromatography.

FIG. 11 is a set of graphs showing the assessment of the competition ofmutants of the humanized anti-NR10 antibody of which the isoelectricpoint of the variable regions is lowered without significant loss of thebinding to NR10, with IL-31.

FIG. 12 shows the effect of the constant region of anti-IL-6 receptorantibody on the heterogeneity assessed by cation exchangechromatography.

FIG. 13 shows the effect of the constant region of anti-IL-6 receptorantibody on the denaturation peak assessed by DSC.

FIG. 14 shows the effect of the novel constant region M14 on theheterogeneity in an anti-IL-6 receptor antibody, assessed by cationexchange chromatography.

FIG. 15 shows the effect of the novel constant region M58 on theheterogeneity in an anti-IL-6 receptor antibody, assessed by cationexchange chromatography.

FIG. 16 shows the effect of the novel constant region M58 on thedenaturation peak in an anti-IL-6 receptor antibody, assessed by DSC.

FIG. 17 shows the result of assaying the retention of huPM1-IgG1 andhuPM1-M58 in the plasma of human FcRn transgenic mice.

FIG. 18 shows the biological activity of each antibody assessed usingBaF/NR10.

FIG. 19 shows the analysis of thermally-accelerated (dotted line) andnon-accelerated (solid line) samples of each modified antibody by cationexchange chromatography to compare the generation of degradationproducts between before and after thermal acceleration. Arrow indicatesthe peak position of basic component which was altered.

FIG. 20 is a set of graphs showing the assessment (BaF) of the activityof each variant.

FIG. 21 is a graph showing the assessment (BaF) of the activity ofHa401La402 and H0L0.

FIG. 22 is a graph showing the assessment (BaF) of the activity ofH17L11 and H0L0.

FIG. 23 is a graph showing the assessment (BaF) of the activity ofH19L12 and H0L0.

FIG. 24 is a graph showing the biological activity of H0L12 and H0L17assessed using BaF/NR10.

FIG. 25-1 is a set of graphs showing the assessment (BaF) of theactivity of each variant.

FIG. 25-2 is a continuation of FIG. 25-1.

FIG. 26 is a schematic diagram for human/mouse wild-type and chimericNR10-ECD.

FIG. 27 is a set of photographs showing the detection of the bindingdomain by Western blotting. A is a photograph showing the result ofdetection using a humanized anti-human NR10 antibody; B is a photographshowing the result of detection using a mouse anti-human NR10 antibody;and C is a photograph showing the result of detection using an anti-Mycantibody. With the anti-human NR10 antibody a binding antigen wasdetected only in hhh, hhm, and hmm, but not in mmm, mmh, and mhm.

FIG. 28-1 shows the amino acid sequence of each variant of H0 (SEQ IDNO: 50).

FIG. 28-2 is a continuation of FIG. 28-1.

FIG. 28-3 is a continuation of FIG. 28-2.

FIG. 29-1 shows the amino acid sequence of each variant of L0 (SEQ IDNO: 52).

FIG. 29-2 is a continuation of FIG. 29-1.

MODE FOR CARRYING OUT THE INVENTION NR10

NR10 is a protein that forms a heterodimer with oncostatin M receptor(OSMR) and functions as an IL-31 receptor. NR10 is also known as glm-r(J Biol Chem 277, 16831-6, 2002), GPL (J Biol Chem 278, 49850-9, 2003),IL31RA (Nat Immunol 5, 752-60, 2004), and such. Thus, NR10 in thepresent invention also includes proteins called by such names.

In the present invention, NR10 (also referred to as IL31RA, GPL, orglm-r) is not particularly limited in terms of its origin, and includesthose derived from humans, mice, monkeys, and other mammals. NR10derived from humans, mice, and monkeys is preferred, and human-derivedNR10 is particularly preferred.

There are multiple known splicing variants of human-derived NR10 (WO00/075314). Of the above-described splicing variants, NR10.1 consists of662 amino acids and contains a transmembrane domain. NR10.2 is a solublereceptor-like protein consisting of 252 amino acids without thetransmembrane domain. Meanwhile, known NR10 splicing variants thatfunction as transmembrane receptor proteins include NR10.3 andIL-31RAv3. The human NR10 of the present invention is not particularlylimited, as long as it forms a heterodimer with oncostatin M receptor(OSMR) and functions as an IL-31 receptor. Preferred NR10 includesNR10.3 (also referred to as ILRAv4 (Nat Immunol 5, 752-60, 2004)) andIL-31RAv3. NR 10.3 (IL31RAv4) consists of 662 amino acids (WO 00/075314;Nat Immunol 5, 752-60, 2004) and IL31RAv3 consists of 732 amino acids(GenBank Accession No: NM_139017). The amino acid sequence of IL31RAv4is shown in SEQ ID NO: 79, and the amino acid sequence of IL31RAv3 isshown in SEQ ID NO: 80. Meanwhile, mouse-derived NR10 includes proteinscomprising the amino acid sequence of SEQ ID NO: 81. In addition,cynomolgus monkey-derived NR10 includes proteins comprising the aminoacid sequence of SEQ ID NO: 66.

Antibodies (Sequences)

Preferred embodiments of the anti-NR10 antibody of the present inventioninclude the anti-NR10 antibodies of any one of (1) to (8) in (A) to (D)below.

(A)NS18

(1) antibodies having a heavy chain variable region that comprises CDR1having the amino acid sequence of SEQ ID NO: 1 (HCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 2 (HCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 3 (HCDR3);(2) antibodies having the heavy chain variable region of SEQ ID NO: 4(VH);(3) antibodies having a light chain variable region that comprises CDR1having the amino acid sequence of SEQ ID NO: 5 (LCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 6 (LCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 7 (LCDR3);(4) antibodies having the light chain variable region of SEQ ID NO: 8(VL);(5) antibodies having the heavy chain variable region of (1) and thelight chain variable region of (3);(6) antibodies having the heavy chain variable region of (2) and thelight chain variable region of (4);(7) antibodies in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibodies of any one of (1) to(6), which have an activity equivalent to that of the antibodies of anyone of (1) to (6); and(8) antibodies that bind to the same epitope as an epitope bound by theantibodies of any one of (1) to (7).

(B) NS22

(1) antibodies having a heavy chain variable region that comprises CDR1having the amino acid sequence of SEQ ID NO: 9 (HCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 10 (HCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 11 (HCDR3);(2) antibodies having the heavy chain variable region of SEQ ID NO: 12(VH);(3) antibodies having a light chain variable region that comprises CDR1having the amino acid sequence of SEQ ID NO: 13 (LCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 14 (LCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 15 (LCDR3);(4) antibodies having the light chain variable region of SEQ ID NO: 16(VL);(5) antibodies having the heavy chain variable region of (1) and thelight chain variable region of (3);(6) antibodies having the heavy chain variable region of (2) and thelight chain variable region of (4);(7) antibodies in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibodies of any one of (1) to(6), which have an activity equivalent to that of the antibodies of anyone of (1) to (6); and(8) antibodies that bind to the same epitope as an epitope bound by theantibodies of any one of (1) to (7).

Specific examples of the above-described substitution, deletion,addition, and/or insertion of one or more amino acids are notparticularly limited and include, for example, the followingmodifications.

Substitution of Ile at position 3 in the heavy chain CDR1 of SEQ ID NO:9 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Val.

Substitution of Met at position 4 in the heavy chain CDR1 of SEQ ID NO:9 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Ile.

Substitution of Met at position 4 in the heavy chain CDR1 of SEQ ID NO:9 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Leu.

Substitution of Ile at position 3 in the heavy chain CDR1 of SEQ ID NO:9 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Ala.

Substitution of Leu at position 1 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Glu.

Substitution of Asn at position 3 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Gln at position 13 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Lys at position 14 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Gln.

Substitution of Lys at position 16 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Gln.

Substitution of Gly at position 17 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Lys and Gly at positions 16 and 17, respectively, in theheavy chain CDR2 of SEQ ID NO: 10 with another amino acid. The aminoacid after substitution is not particularly limited but preferredexamples include substitution of Lys at position 16 with Gln, and Gly atposition 17 with Asp.

Substitution of Lys, Lys, and Gly at positions 14, 16, and 17,respectively, in the heavy chain CDR2 of SEQ ID NO: 10 with anotheramino acid. The amino acid after substitution is not particularlylimited but preferred examples include substitution of Lys at position14 with Gln, Lys at position 16 with Gln, and Gly at position 17 withAsp.

Substitution of Gln, Lys, Lys, and Gly at positions 13, 14, 16, and 17,respectively, in the heavy chain CDR2 of SEQ ID NO: 10 with anotheramino acid. The amino acid after substitution is not particularlylimited but preferred examples include substitution of Gln at position13 with Asp, Lys at position 14 with Gln, Lys at position 16 with Gln,and Gly at position 17 with Asp.

Substitution of Ser at position 10 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Gln at position 13 in the heavy chain CDR2 of SEQ ID NO:10 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Pro.

Substitution of Tyr at position 3 in the heavy chain CDR3 of SEQ ID NO:11 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Leu.

Substitution of Met at position 10 in the heavy chain CDR3 of SEQ ID NO:11 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Leu.

Substitution of Asp at position 11 in the heavy chain CDR3 of SEQ ID NO:11 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Glu.

Substitution of Tyr at position 12 in the heavy chain CDR3 of SEQ ID NO:11 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Thr and Ser.

Substitution of Met, Asp, and Tyr at positions 10, 11, and 12,respectively, in the heavy chain CDR3 of SEQ ID NO: 11 with anotheramino acid. The amino acid after substitution is not particularlylimited but preferred examples include substitution of Met at position10 with Leu, Asp at position 11 with Glu, and Tyr at position 12 withThr.

Substitution of Asp and Tyr at positions 11 and 12, respectively, in theheavy chain CDR3 of SEQ ID NO: 11 with another amino acid. The aminoacid after substitution is not particularly limited but preferredexamples include substitution of Asp at position 11 with Glu, and Tyr atposition 12 with Thr.

Substitution of Tyr, Asp, and Tyr at positions 3, 11, and 12,respectively, in the heavy chain CDR3 of SEQ ID NO: 11 with anotheramino acid. The amino acid after substitution is not particularlylimited but preferred examples include substitution of Tyr at position 3with Leu, Asp at position 11 with Glu, and Tyr at position 12 with Thror Ser.

Substitution of Arg at position 1 in the light chain CDR1 of SEQ ID NO:13 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Gln.

Substitution of Asn at position 5 in the light chain CDR1 of SEQ ID NO:13 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Arg and Asn at positions 1 and 5, respectively, in thelight chain CDR1 of SEQ ID NO: 13 with another amino acid. The aminoacid after substitution is not particularly limited but preferredexamples include substitution of Arg at position 1 with Gln, and Asn atposition 5 with Asp.

Substitution of Ser at position 8 in the light chain CDR1 of SEQ ID NO:13 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Arg.

Substitution of Leu at position 10 in the light chain CDR1 of SEQ ID NO:13 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Val.

Substitution of Ser and Leu at positions 8 and 10, respectively, in thelight chain CDR1 of SEQ ID NO: 13 with another amino acid. The aminoacid after substitution is not particularly limited but preferredexamples include substitution of Ser at position 8 with Arg, and Leu atposition 10 with Val.

Substitution of Thr at position 2 in the light chain CDR1 of SEQ ID NO:13 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Ala and Ser.

Substitution of Asn at position 1 in the light chain CDR2 of SEQ ID NO:14 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Lys at position 3 in the light chain CDR2 of SEQ ID NO:14 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Gln.

Substitution of Leu at position 5 in the light chain CDR2 of SEQ ID NO:14 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Glu.

Substitution of Lys at position 7 in the light chain CDR2 of SEQ ID NO:14 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Gln and Asp.

Substitution of Lys, Leu, and Lys at positions 3, 5, and 7,respectively, in the light chain CDR2 of SEQ ID NO: 14 with anotheramino acid. The amino acid after substitution is not particularlylimited but preferred examples include substitution of Lys at position 3with Gln, Leu at position 5 with Glu, and Lys at position 7 with Gln.

Substitution of Glu at position 5 in the light chain CDR3 of SEQ ID NO:15 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Ser at position 6 in the light chain CDR3 of SEQ ID NO:15 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Asp.

Substitution of Thr at position 9 in the light chain CDR3 of SEQ ID NO:15 with another amino acid. The amino acid after substitution is notparticularly limited but preferred examples thereof include Phe.

Each of the above-described substitutions may be made alone, or multiplesubstitutions may be made in combination. Furthermore, the abovesubstitutions may be combined with other substitutions. Thesesubstitutions can improve the antibody pharmacokinetics (retention inplasma), enhance the antigen-binding activity, improve the stability,and/or reduce the risk of immunogenicity.

In the present invention, specific examples of the variable regionshaving a combination of the above-described substitutions include, forexample, heavy chain variable regions having the amino acid sequence ofSEQ ID NO: 167 and light chain variable regions having the amino acidsequence of SEQ ID NO: 168. Moreover, examples of the antibodies havinga combination of the above-described substitutions include, for example,antibodies that comprise a heavy chain variable region having the aminoacid sequence of SEQ ID NO: 167 and a light chain variable region havingthe amino acid sequence of SEQ ID NO: 168.

Moreover, specific examples of the heavy chain or light chain variableregions having a combination of the above-described substitutionsinclude, for example, the following variable regions:

(a) heavy chain variable regions that comprise CDR1 of SEQ ID NO: 196,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 11 (H17);(b) heavy chain variable regions that comprise CDR1 of SEQ ID NO: 176,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 11 (H19);(c) heavy chain variable regions that comprise CDR1 of SEQ ID NO: 196,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 184 (H28, H42);(d) heavy chain variable regions that comprises CDR1 of SEQ ID NO: 9,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 184 (H30, H44);(e) heavy chain variable regions that comprise CDR1 of SEQ ID NO: 176,CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 184 (H34, H46);(f) heavy chain variable regions that comprise CDR1 of SEQ ID NO: 9,CDR2 of SEQ ID NO: 198, and CDR3 of SEQ ID NO: 184 (H57, H78);(g) heavy chain variable regions that comprise CDR1 of SEQ ID NO: 176,CDR2 of SEQ ID NO: 198, and CDR3 of SEQ ID NO: 184 (H71, H92);(h) heavy chain variable regions that comprise CDR1 of SEQ ID NO: 9,CDR2 of SEQ ID NO: 199, and CDR3 of SEQ ID NO: 184 (H97, H98);(i) light chain variable regions that comprise CDR1 of SEQ ID NO: 200,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L11);(j) light chain variable regions that comprise CDR1 of SEQ ID NO: 201,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L12);(k) light chain variable regions that comprise CDR1 of SEQ ID NO: 202,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L17); and(l) light chain variable regions that comprise CDR1 of SEQ ID NO: 203,CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L50).

Furthermore, specific examples of the antibodies having a combination ofthe above-described substitutions include, for example:

(i) antibodies that comprise the heavy chain variable region of (c) andthe light chain variable region of (k);(ii) antibodies that comprise the heavy chain variable region of (d) andthe light chain variable region of (k);(iii) antibodies that comprise the heavy chain variable region of (e)and the light chain variable region of (k);(iv) antibodies that comprise the heavy chain variable region of (f) andthe light chain variable region of (k);(v) antibodies that comprise the heavy chain variable region of (g) andthe light chain variable region of (k); and(vi) antibodies that comprise the heavy chain variable region of (h) andthe light chain variable region of (1).

(C) NS23

(1) antibodies having a heavy chain variable region that comprises CDR1having the amino acid sequence of SEQ ID NO: 17 (HCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 18 (HCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 19 (HCDR3);(2) antibodies having the heavy chain variable region of SEQ ID NO: 20(VH);(3) antibodies having a light chain variable region that comprises CDR1having the amino acid sequence of SEQ ID NO: 21 (LCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 22 (LCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 23 (LCDR3);(4) antibodies having the light chain variable region of SEQ ID NO: 24(VL);(5) antibodies having the heavy chain variable region of (1) and thelight chain variable region of (3);(6) antibodies having the heavy chain variable region of (2) and thelight chain variable region of (4);(7) antibodies in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibodies of any one of (1) to(6), which have an activity equivalent to that of the antibodies of anyone of (1) to (6); and(8) antibodies that bind to the same epitope as an epitope bound by theantibodies of any one of (1) to (7).

(D) NS33

(1) antibodies having a heavy chain variable region that comprise CDR1having the amino acid sequence of SEQ ID NO: 25 (HCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 26 (HCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 27 (HCDR3);(2) antibodies having the heavy chain variable region of SEQ ID NO: 28(VH);(3) antibodies having a light chain variable region that comprise CDR1having the amino acid sequence of SEQ ID NO: 29 (LCDR1), CDR2 having theamino acid sequence of SEQ ID NO: 30 (LCDR2), and CDR3 having the aminoacid sequence of SEQ ID NO: 31 (LCDR3);(4) antibodies having the light chain variable region of SEQ ID NO: 32(VL);(5) antibodies having the heavy chain variable region of (1) and thelight chain variable region of (3);(6) antibodies having the heavy chain variable region of (2) and thelight chain variable region of (4);(7) antibodies in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibodies of any one of (1) to(6), which have an activity equivalent to that of the antibodies of anyone of (1) to (6); and(8) antibodies that bind to the same epitope as an epitope bound by theantibodies of any one of (1) to (7).

Any framework regions (FR) may be used for the above-describedantibodies of (1) or (3); however, FRs derived from human are preferablyused. Furthermore, any constant regions may be used for theabove-described antibodies of (1) to (8); however, constant regionsderived from human are preferably used. For the antibodies of thepresent invention, the amino acid sequence of the original FR orconstant region may be used without modification, or after beingmodified to a different amino acid sequence by substitution, deletion,addition, and/or insertion of one or more amino acids.

The amino acid sequence of the heavy chain of the above-described NS18is shown in SEQ ID NO: 34 and the nucleotide sequence encoding thisamino acid sequence is shown in SEQ ID NO: 33. Meanwhile, the amino acidsequence of the light chain is shown in SEQ ID NO: 36 and the nucleotidesequence encoding this amino acid sequence is shown in SEQ ID NO: 35.

The amino acid sequence of the heavy chain of NS22 is shown in SEQ IDNO: 38 and the nucleotide sequence encoding this amino acid sequence isshown in SEQ ID NO: 37. Meanwhile, the amino acid sequence of the lightchain is shown in SEQ ID NO: 40 and the nucleotide sequence encodingthis amino acid sequence is shown in SEQ ID NO: 39.

The amino acid sequence of the heavy chain of NS23 is shown in SEQ IDNO: 42 and the nucleotide sequence encoding this amino acid sequence isshown in SEQ ID NO: 41. Meanwhile, the amino acid sequence of the lightchain is shown in SEQ ID NO: 44 and the nucleotide sequence encodingthis amino acid sequence is shown in SEQ ID NO: 43.

The amino acid sequence of the heavy chain of NS33 is shown in SEQ IDNO: 46 and the nucleotide sequence encoding this amino acid sequence isshown in SEQ ID NO: 45. Meanwhile, the amino acid sequence of the lightchain is shown in SEQ ID NO: 48 and the nucleotide sequence encodingthis amino acid sequence is shown in SEQ ID NO: 47.

In the present invention, the “activity equivalent to that of theantibody of any one of (1) to (6)” means that the activity of bindingand/or neutralizing NR10 (for example, human NR10) is equivalent. In thepresent invention, the term “equivalent” means that the activity is notnecessarily the same but may be enhanced or reduced as long as theactivity is retained. Antibodies with a reduced activity include, forexample, antibodies having an activity that is 30% or more, preferably50% or more, and more preferably 80% or more of that of the originalantibody.

The antibodies of any one of (1) to (6) mentioned above may have asubstitution, deletion, addition, and/or insertion of one or more aminoacids in the amino acid sequence of the variable regions (CDR sequencesand/or FR sequences), as long as the NR10-binding and/or neutralizingactivity is retained. Methods well known to those skilled in the art toprepare the amino acid sequence of an antibody that has a substitution,deletion, addition, and/or insertion of one or more amino acids in theamino acid sequence and retains NR10-binding and/or neutralizingactivity, include methods for introducing mutations into proteins. Forexample, those skilled in the art can prepare mutants functionallyequivalent to the antibody having NR10-binding and/or neutralizingactivity by introducing appropriate mutations into the amino acidsequence of the antibody having NR10-binding and/or neutralizingactivity using site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T,Ogasahara, Y, and Nakagawa, M. (1995) Anoligodeoxyribonucleotide-directed dual amber method for site-directedmutagenesis. Gene 152, 271-275, Zoller, M J, and Smith, M. (1983)Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors. Methods Enzymol. 100, 468-500, Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, H J (1984) The gapped duplexDNA approach to oligonucleotide-directed mutation construction. NucleicAcids Res. 12, 9441-9456, Kramer W, and Fritz H J (1987)Oligonucleotide-directed construction of mutations via gapped duplex DNAMethods. Enzymol. 154, 350-367, Kunkel, TA (1985) Rapid and efficientsite-specific mutagenesis without phenotypic selection. Proc Natl AcadSci USA. 82, 488-492) or the like. Thus, antibodies that contain one ormore amino acid mutations in the variable regions and have NR10-bindingand/or neutralizing activity are also included in the antibody of thepresent invention.

When an amino acid residue is altered, the amino acid is preferablymutated for a different amino acid(s) that conserves the properties ofthe amino acid side-chain. Examples of amino acid side chain propertiesare: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T), aminoacids containing aliphatic side chains (G, A, V, L, I, and P), aminoacids containing hydroxyl group-containing side chains (S, T, and Y),amino acids containing sulfur-containing side chains (C and M), aminoacids containing carboxylic acid- and amide-containing side chains (D,N, E, and Q), amino acids containing basic side chains (R, K, and H),and amino acids containing aromatic side chains (H, F, Y, and W) (aminoacids are represented by one-letter codes in parentheses). Amino acidsubstitutions within each group are called conservative substitutions.It is already known that a polypeptide containing a modified amino acidsequence in which one or more amino acid residues in a given amino acidsequence are deleted, added, and/or substituted with other amino acidscan retain the original biological activity (Mark, D. F. et al., Proc.Natl. Acad. Sci. USA; (1984) 81:5662-6; Zoller, M. J. and Smith, M.,Nucleic Acids Res. (1982) 10:6487-500; Wang, A. et al., Science (1984)224:1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA(1982) 79:6409-13). Such mutants have an amino acid identity of at least70%, more preferably at least 75%, even more preferably at least 80%,still more preferably at least 85%, yet more preferably at least 90%,and most preferably at least 95%, with the variable regions (forexample, CDR sequences, FR sequences, or whole variable regions) of thepresent invention. Herein, sequence identity is defined as thepercentage of residues identical to those in the original amino acidsequence of the heavy chain variable region or light chain variableregion, determined after the sequences are aligned and gaps areappropriately introduced to maximize the sequence identity as necessary.The identity of amino acid sequences can be determined by the methoddescribed below.

Alternatively, the amino acid sequences of variable regions that have asubstitution, deletion, addition, and/or insertion of one or more aminoacids in the amino acid sequence of the variable regions (CDR sequencesand/or FR sequences) and retain NR10-binding and/or neutralizingactivity can be obtained from nucleic acids that hybridize understringent conditions to nucleic acid composed of the nucleotide sequenceencoding the amino acid sequence of the variable regions. Stringenthybridization conditions to isolate a nucleic acid that hybridizes understringent conditions to a nucleic acid that includes the nucleotidesequence encoding the amino acid sequence of the variable regionsinclude, for example, the conditions of 6M urea, 0.4% SDS, 0.5×SSC, and37° C., or hybridization conditions with stringencies equivalentthereto. With more stringent conditions, for example, the conditions of6M urea, 0.4% SDS, 0.1×SSC, and 42° C., isolation of nucleic acids witha much higher homology can be expected. The sequences of the isolatednucleic acids can be determined by the known methods described below.The overall nucleotide sequence homology of the isolated nucleic acid isat least 50% or higher sequence identity, preferably 70% or higher, morepreferably 90% or higher (for example, 95%, 96%, 97%, 98%, 99%, orhigher).

Nucleic acids that hybridize under stringent conditions to a nucleicacid composed of the nucleotide sequence encoding the amino acidsequence of the variable regions can also be isolated using, instead ofthe above-described methods using hybridization techniques, geneamplification methods such as polymerase chain reaction (PCR) usingprimers synthesized based on the information of nucleotide sequenceencoding the amino acid sequence of the variable regions.

Specifically, the identity of one nucleotide sequence or amino acidsequence to another can be determined using the algorithm BLAST, byKarlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90, 5873-7).Programs such as BLASTN and BLASTX were developed based on thisalgorithm (Altschul et al., J. Mol. Biol. (1990) 215, 403-10). Toanalyze nucleotide sequences according to BLASTN based on BLAST, theparameters are set, for example, as score=100 and wordlength=12. On theother hand, parameters used for the analysis of amino acid sequences byBLASTX based on BLAST include, for example, score=50 and wordlength=3.Default parameters for each program are used when using the BLAST andGapped BLAST programs. Specific techniques for such analyses are knownin the art (see the website of the National Center for BiotechnologyInformation (NCBI), Basic Local Alignment Search Tool (BLAST);http://www.ncbi.nlm.nih.gov).

The present invention also provides antibodies that bind to the sameepitope as an epitope bound by the antibodies of any one of (1) to (7).

Whether an antibody recognizes the same epitope as that recognized byanother antibody can be confirmed by the competition between the twoantibodies against the epitope. Competition between the antibodies canbe evaluated by competitive binding assays using means such as ELISA,fluorescence energy transfer method (FRET), and fluorometric microvolumeassay technology (FMAT®). The amount of antibodies bound to an antigenindirectly correlate with the binding ability of candidate competitorantibodies (test antibodies) that competitively bind to the sameepitope. In other words, as the amount of or the affinity of testantibodies against the same epitope increases, the amount of antibodiesbound to the antigen decreases, and the amount of test antibodies boundto the antigen increases. Specifically, appropriately labeled antibodiesand antibodies to be evaluated are simultaneously added to the antigens,and the thus bound antibodies are detected using the label. The amountof antibodies bound to the antigen can be easily determined by labelingthe antibodies beforehand. This label is not particularly limited, andthe labeling method is selected according to the assay technique used.The labeling method includes fluorescent labeling, radiolabeling,enzymatic labeling, and such.

For example, fluorescently labeled antibodies and unlabeled antibodiesor test antibodies are simultaneously added to animal cells expressingNR10, and the labeled antibodies are detected by fluorometricmicrovolume assay technology.

Herein, the “antibody that recognizes the same epitope” refers to anantibody that can reduce the binding of the labeled antibody by at least50% at a concentration that is usually 100 times higher, preferably 80times higher, more preferably 50 times higher, even more preferably 30times higher, and still more preferably 10 times higher than aconcentration at which the non-labeled antibody reduces the binding ofthe labeled antibody by 50% (IC₅₀).

Antibodies that bind to the epitope to which the antibodies set forth inany one of (1) to (7) above bind are useful because they have aparticularly high neutralizing activity.

The antibodies set forth in any one of (1) to (8) above are preferablyhumanized antibodies, but are not particularly limited thereto.

Furthermore, the present invention provides genes encoding the anti-NR10antibodies of any one of (1) to (8) of (A) to (D) above. The genes ofthe present invention may be any form of genes, for example, DNAs orRNAs.

Antibodies (Humanized)

Preferred embodiments of the antibodies of the present invention includehumanized antibodies that bind to NR10. The humanized antibodies can beprepared by methods known to those skilled in the art.

The variable region of antibody is typically composed of threecomplementarity-determining regions (CDRs) sandwiched by four frames(FRs). The CDRs substantially determine the binding specificity ofantibody. The amino acid sequences of CDRs are highly diverse. Incontrast, the amino acid sequences of FRs often exhibit high homologybetween antibodies having different binding specificities. It istherefore said in general that the binding specificity of an antibodycan be transplanted to a different antibody by grafting the CDRs.

Humanized antibodies are also referred to as reshaped human antibodies,and they are prepared by transferring the CDRs of an antibody derivedfrom a non-human mammal such as a mouse, to the CDRs of a humanantibody. General genetic recombination techniques for their preparationare also known (see European Patent Application Publication No. 125023and WO 96/02576).

Specifically, for example, when the CDRs are derived from a mouseantibody, a DNA sequence designed such that the CDRs of the mouseantibody are linked with framework regions (FRs) of human antibody issynthesized by PCR using, as primers, several oligonucleotides that haveportions overlapping the ends of both CDRs and FRs (see the methoddescribed in WO 98/13388). The resulting DNA is then ligated to a DNAencoding a human antibody constant region, inserted into an expressionvector, and introduced into a host to produce the antibody (see EuropeanPatent Application Publication No. EP 239400 and International PatentApplication Publication No. WO 96/02576).

Human antibody framework regions to be linked with CDRs are selected sothat the CDRs form a favorable antigen-binding site. If needed, aminoacid substitution, deletion, addition, and/or insertion may beintroduced into the framework regions of antibody variable region sothat the CDRs of the reshaped human antibody form a properantigen-binding site. For example, mutations can be introduced into theamino acid sequence of FR by applying the PCR method which is used tograft mouse CDRs to human FRs. Specifically, mutations can be introducedinto a portion of the nucleotide sequences of primers that anneal to theFRs. The mutations are introduced into FRs synthesized by such primers.The antigen-binding activity of mutant antibodies having amino acidsubstitutions can be determined and assessed by the method describedabove, and thereby mutant FR sequences having desired properties can beselected (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

Constant (C) regions from human antibodies are used for those ofhumanized antibodies. For example, Cγ1, Cγ2, Cγ3, Cγ4, Cμ, Cδ, Cα1, Cα2,and Cε are used for H chains; and Cκ and Cλ are used for L chains. Theamino acid sequence of Cκ is shown in SEQ ID NO: 58, and the nucleotidesequence encoding this amino acid sequence is shown in SEQ ID NO: 57.The amino acid sequence of Cγ1 is shown in SEQ ID NO: 60, and thenucleotide sequence encoding this amino acid sequence is shown in SEQ IDNO: 59. The amino acid sequence of Cγ2 is shown in SEQ ID NO: 62, andthe nucleotide sequence encoding this amino acid sequence is shown inSEQ ID NO: 61. The amino acid sequence of Cγ4 is shown in SEQ ID NO: 64,and the nucleotide sequence encoding this amino acid sequence is shownin SEQ ID NO: 63. Furthermore, human antibody C regions may be modifiedto improve the stability of antibody or antibody production. Modifiedhuman antibody C regions include, for example, the C regions describedherein below. Human antibodies used for humanization may be of anyisotype such as IgG, IgM, IgA, IgE, or IgD; however, IgG is preferablyused in the present invention. IgG that can be used includes IgG1, IgG2,IgG3, IgG4, and the like.

Moreover, after a humanized antibody is prepared, amino acids in thevariable region (for example, CDR and FR) and constant region of thehumanized antibody may be deleted, added, inserted, and/or substitutedwith other amino acids. The antibodies of the present invention alsoinclude such humanized antibodies with amino acid substitutions and thelike.

The origin of CDRs of a humanized antibody is not particularly limited,and may be any animal. For example, it is possible to use the sequencesof mouse antibodies, rat antibodies, rabbit antibodies, camelantibodies, and the like. CDR sequences of mouse antibodies arepreferred.

In general, it is difficult to humanize antibodies while retaining thebinding and neutralizing activities of the original antibodies. Thepresent invention, however, succeeded in obtaining humanized antibodieshaving the binding and/or neutralizing activities equivalent to those ofthe original mouse antibodies. Humanized antibodies are useful whenadministered to humans for the therapeutic purposes, because theyexhibit reduced immunogenicity in the human body.

Preferred examples of the humanized anti-NR10 antibodies of the presentinvention include, for example:

(a) humanized antibodies that comprise a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 50 (H0-VH);(b) humanized antibodies that comprise a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 112 (H1-VH);(c) humanized antibodies that comprise a light chain variable regionhaving the amino acid sequence of SEQ ID NO: 52 (L0-VL);(d) humanized antibodies that comprise a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 50 (H0-VH) and a lightchain variable region having the amino acid sequence of SEQ ID NO: 52(L0-VL); and(e) humanized antibodies that comprise a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 112 and a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

The heavy chain variable region having the amino acid sequence of SEQ IDNO: 50 (H0-VH), heavy chain variable region having the amino acidsequence of SEQ ID NO: 112, and light chain variable region having theamino acid sequence of SEQ ID NO: 52 (L0-VL) may have a substitution,deletion, addition, and/or insertion of one or more amino acids. Thesubstitution, deletion, addition, and/or insertion of amino acids may bemade in either or both of the CDRs and FRs.

Thus, other preferred embodiments of the humanized anti-NR10 antibody ofthe present invention include, for example:

(f) antibodies that comprise a heavy chain variable region having anamino acid sequence in which one or more amino acids are substituted,deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO:50 (H0-VH);(g) antibodies that comprise a heavy chain variable region having anamino acid sequence in which one or more amino acids are substituted,deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO:112 (H1-VH);(h) antibodies that comprise a light chain variable region having anamino acid sequence in which one or more amino acids are substituted,deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO:52 (L0-VL);(i) antibodies that comprise a heavy chain variable region having anamino acid sequence in which one or more amino acids are substituted,deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO:50 (H0-VH), and a light chain variable region having an amino acidsequence in which one or more amino acids are substituted, deleted,added, and/or inserted in the amino acid sequence of SEQ ID NO: 52(L0-VL);(j) antibodies that comprise a heavy chain variable region having anamino acid sequence in which one or more amino acids are substituted,deleted, added, and/or inserted in the amino acid sequence of SEQ ID NO:112 (H1-VH), and a light chain variable region having an amino acidsequence in which one or more amino acids are substituted, deleted,added, and/or inserted in the amino acid sequence of SEQ ID NO: 52(L0-VL);

Without particular limitation, the antibodies of any one of (f) to (j)preferably have an activity similar to that of the antibodies of any oneof (a) to (e).

The substitution, deletion, addition, and/or insertion of amino acidsare not particularly limited, but specific examples include, forexample, the above-described amino acid substitutions.

More specifically, for example, the following amino acid substitutionsmay be included:

Substitution of Ile at position 3 of CDR1 (SEQ ID NO: 9) in the heavychain variable region of SEQ ID NO: 50 or 112 with Val (SEQ ID NO: 173).Thus, the present invention provides heavy chain variable regions inwhich CDR1 having the amino acid sequence of SEQ ID NO: 9 is substitutedwith CDR1 having the amino acid sequence of SEQ ID NO: 173 in a heavychain variable region having the amino acid sequence of SEQ ID NO: 50 or112.

Substitution of Met at position 4 of CDR1 (SEQ ID NO: 9) in the heavychain variable region of SEQ ID NO: 50 or 112 with Ile (SEQ ID NO: 174).Thus, the present invention provides heavy chain variable regions inwhich CDR1 having the amino acid sequence of SEQ ID NO: 9 is substitutedwith CDR1 having the amino acid sequence of SEQ ID NO: 174 in a heavychain variable region having the amino acid sequence of SEQ ID NO: 50 or112.

Substitution of Met at position 4 of CDR1 (SEQ ID NO: 9) in the heavychain variable region of SEQ ID NO: 50 or 112 with Leu (SEQ ID NO: 175).Thus, the present invention provides heavy chain variable regions inwhich CDR1 having the amino acid sequence of SEQ ID NO: 9 is substitutedwith CDR1 having the amino acid sequence of SEQ ID NO: 175 in a heavychain variable region having the amino acid sequence of SEQ ID NO: 50 or112.

Substitution of Ile at position 3 of CDR1 (SEQ ID NO: 9) in the heavychain variable region of SEQ ID NO: 50 or 112 with Ala (SEQ ID NO: 176).Thus, the present invention provides heavy chain variable regions inwhich CDR1 having the amino acid sequence of SEQ ID NO: 9 is substitutedwith CDR1 having the amino acid sequence of SEQ ID NO: 176 in a heavychain variable region having the amino acid sequence of SEQ ID NO: 50 or112.

Substitution of Leu at position 1 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Glu (SEQ ID NO: 113).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 113in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Asn at position 3 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Asp (SEQ ID NO: 114).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 114in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Gln at position 13 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Asp (SEQ ID NO: 115).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 115in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Lys at position 14 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Gln (SEQ ID NO: 116).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 116in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Lys at position 16 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Gln (SEQ ID NO: 117).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 117in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Gly at position 17 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Asp (SEQ ID NO: 118).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 118in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Lys at position 16 and Gly at position 17 of CDR2 (SEQID NO: 10) in the heavy chain variable region of SEQ ID NO: 50 or 112with Gln and Asp, respectively (SEQ ID NO: 119). Thus, the presentinvention provides heavy chain variable regions in which CDR2 having theamino acid sequence of SEQ ID NO: 10 is substituted with CDR2 having theamino acid sequence of SEQ ID NO: 119 in a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 50 or 112.

Substitution of Lys at position 14, Lys at position 16, and Gly atposition 17 of CDR2 (SEQ ID NO: 10) in the heavy chain variable regionof SEQ ID NO: 50 or 112 with Gln, Gln, and Asp, respectively (SEQ ID NO:167). Thus, the present invention provides heavy chain variable regionsin which CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 171in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Gln at position 13, Lys at position 14, Lys at position16, and Gly at position 17 of CDR2 (SEQ ID NO: 10) in the heavy chainvariable region of SEQ ID NO: 50 or 112 with Asp, Gln, Gln, and Asp,respectively (SEQ ID NO: 172). Thus, the present invention providesheavy chain variable regions in which CDR2 having the amino acidsequence of SEQ ID NO: 10 is substituted with CDR2 having the amino acidsequence of SEQ ID NO: 172 in a heavy chain variable region having theamino acid sequence of SEQ ID NO: 50 or 112.

Substitution of Ser at position 10 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Asp (SEQ ID NO: 177).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 177in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Gln at position 13 of CDR2 (SEQ ID NO: 10) in the heavychain variable region of SEQ ID NO: 50 or 112 with Pro (SEQ ID NO: 178).Thus, the present invention provides heavy chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 10 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 178in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Tyr at position 3 of CDR3 (SEQ ID NO: 11) in the heavychain variable region of SEQ ID NO: 50 or 112 with Leu (SEQ ID NO: 179).Thus, the present invention provides heavy chain variable regions inwhich CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 179in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Met at position 10 of CDR3 (SEQ ID NO: 11) in the heavychain variable region of SEQ ID NO: 50 or 112 with Leu (SEQ ID NO: 180).Thus, the present invention provides heavy chain variable regions inwhich CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 180in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Asp at position 11 of CDR3 (SEQ ID NO: 11) in the heavychain variable region of SEQ ID NO: 50 or 112 with Glu (SEQ ID NO: 181).Thus, the present invention provides heavy chain variable regions inwhich CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 181in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Tyr at position 12 of CDR3 (SEQ ID NO: 11) in the heavychain variable region of SEQ ID NO: 50 or 112 with Thr (SEQ ID NO: 182).Thus, the present invention provides heavy chain variable regions inwhich CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 182in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Tyr at position 12 of CDR3 (SEQ ID NO: 11) in the heavychain variable region of SEQ ID NO: 50 or 112 with Ser (SEQ ID NO: 183).Thus, the present invention provides heavy chain variable regions inwhich CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 183in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Met at position 10, Asp at position 11, and Tyr atposition 12 of CDR3 (SEQ ID NO: 11) in the heavy chain variable regionof SEQ ID NO: 50 or 112 with Leu, Glu, Thr, respectively (SEQ ID NO:184). Thus, the present invention provides heavy chain variable regionsin which CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 184in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Asp at position 11 and Tyr at position 12 of CDR3 (SEQID NO: 11) in the heavy chain variable region of SEQ ID NO: 50 or 112with Glu and Thr, respectively (SEQ ID NO: 185). Thus, the presentinvention provides heavy chain variable regions in which CDR3 having theamino acid sequence of SEQ ID NO: 11 is substituted with CDR3 having theamino acid sequence of SEQ ID NO: 185 in a heavy chain variable regionhaving the amino acid sequence of SEQ ID NO: 50 or 112.

Substitution of Tyr at position 3, Asp at position 11, and Tyr atposition 12 of CDR3 (SEQ ID NO: 11) in the heavy chain variable regionof SEQ ID NO: 50 or 112 with Leu, Glu, and Thr, respectively (SEQ ID NO:186). Thus, the present invention provides heavy chain variable regionsin which CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 186in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Tyr at position 3, Asp at position 11, and Tyr atposition 12 of CDR3 (SEQ ID NO: 11) in the heavy chain variable regionof SEQ ID NO: 50 or 112 with Leu, Glu, and Ser, respectively (SEQ ID NO:187). Thus, the present invention provides heavy chain variable regionsin which CDR3 having the amino acid sequence of SEQ ID NO: 11 issubstituted with CDR3 having the amino acid sequence of SEQ ID NO: 187in a heavy chain variable region having the amino acid sequence of SEQID NO: 50 or 112.

Substitution of Arg at position 1 of CDR1 (SEQ ID NO: 13) in the lightchain variable region of SEQ ID NO: 52 with Gln (SEQ ID NO: 121). Thus,the present invention provides light chain variable regions in whichCDR1 having the amino acid sequence of SEQ ID NO: 13 is substituted withCDR1 having the amino acid sequence of SEQ ID NO: 121 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Asn at position 5 of CDR1 (SEQ ID NO: 13) in the lightchain variable region of SEQ ID NO: 52 with Asp (SEQ ID NO: 122). Thus,the present invention provides light chain variable regions in whichCDR1 having the amino acid sequence of SEQ ID NO: 13 is substituted withCDR1 having the amino acid sequence of SEQ ID NO: 122 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Ser at position 8 of CDR1 (SEQ ID NO: 13) in the lightchain variable region of SEQ ID NO: 52 with Arg (SEQ ID NO: 188). Thus,the present invention provides light chain variable regions in whichCDR1 having the amino acid sequence of SEQ ID NO: 13 is substituted withCDR1 having the amino acid sequence of SEQ ID NO: 188 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Leu at position 10 of CDR1 (SEQ ID NO: 13) of the lightchain variable region of SEQ ID NO: 52 with Val (SEQ ID NO: 189). Thus,the present invention provides light chain variable regions in whichCDR1 having the amino acid sequence of SEQ ID NO: 13 is substituted withCDR1 having the amino acid sequence of SEQ ID NO: 189 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Ser at position 8 and Leu at position 10 of CDR1 (SEQ IDNO: 13) of the light chain variable region of SEQ ID NO: 52 with Arg andVal, respectively (SEQ ID NO: 190). Thus, the present invention provideslight chain variable regions in which CDR1 having the amino acidsequence of SEQ ID NO: 13 is substituted with CDR1 having the amino acidsequence of SEQ ID NO: 190 in a light chain variable region having theamino acid sequence of SEQ ID NO: 52.

Substitution of Thr at position 2 of CDR1 (SEQ ID NO: 13) in the lightchain variable region of SEQ ID NO: 52 with Ala (SEQ ID NO: 191). Thus,the present invention provides light chain variable regions in whichCDR1 having the amino acid sequence of SEQ ID NO: 13 is substituted withCDR1 having the amino acid sequence of SEQ ID NO: 191 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Thr at position 2 of CDR1 (SEQ ID NO: 13) in the lightchain variable region of SEQ ID NO: 52 with Ser (SEQ ID NO: 192). Thus,the present invention provides light chain variable regions in whichCDR1 having the amino acid sequence of SEQ ID NO: 13 is substituted withCDR1 having the amino acid sequence of SEQ ID NO: 192 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Asn at position 1 of CDR2 (SEQ ID NO: 14) in the lightchain variable region of SEQ ID NO: 52 with Asp (SEQ ID NO: 123). Thus,the present invention provides light chain variable regions in whichCDR2 having the amino acid sequence of SEQ ID NO: 14 is substituted withCDR2 having the amino acid sequence of SEQ ID NO: 123 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Lys at position 3 of CDR2 (SEQ ID NO: 14) in the lightchain variable region of SEQ ID NO: 52 with Gln (SEQ ID NO: 124). Thus,the present invention provides light chain variable regions in whichCDR2 having the amino acid sequence of SEQ ID NO: 14 is substituted withCDR2 having the amino acid sequence of SEQ ID NO: 124 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Leu at position 5 of CDR2 (SEQ ID NO: 14) in the lightchain variable region of SEQ ID NO: 52 with Glu (SEQ ID NO: 125). Thus,the present invention provides light chain variable regions in whichCDR2 having the amino acid sequence of SEQ ID NO: 14 is substituted withCDR2 having the amino acid sequence of SEQ ID NO: 125 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Lys at position 7 of CDR2 (SEQ ID NO: 14) in the lightchain variable region of SEQ ID NO: 52 with Gln (SEQ ID NO: 126). Thus,the present invention provides light chain variable regions in whichCDR2 having the amino acid sequence of SEQ ID NO: 14 is substituted withCDR2 having the amino acid sequence of SEQ ID NO: 126 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Lys at position 7 of CDR2 (SEQ ID NO: 14) in the lightchain variable region of SEQ ID NO: 52 with Asp (SEQ ID NO: 127). Thus,the present invention provides light chain variable regions in whichCDR2 having the amino acid sequence of SEQ ID NO: 14 is substituted withCDR2 having the amino acid sequence of SEQ ID NO: 127 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Arg at position 1 and Asn at position 5 of CDR1 (SEQ IDNO: 13) in the light chain variable region of SEQ ID NO: 52 with Gln andAsp, respectively (SEQ ID NO: 169). Thus, the present invention provideslight chain variable regions in which CDR1 having the amino acidsequence of SEQ ID NO: 13 is substituted with CDR1 having the amino acidsequence of SEQ ID NO: 169 in a light chain variable region having theamino acid sequence of SEQ ID NO: 52.

Substitution of Lys at position 3, Leu at position 5, and Lys atposition 7 of CDR2 (SEQ ID NO: 14) in the light chain variable region ofSEQ ID NO: 52 with Gln, Glu, and Gln, respectively (SEQ ID NO: 170).Thus, the present invention provides light chain variable regions inwhich CDR2 having the amino acid sequence of SEQ ID NO: 14 issubstituted with CDR2 having the amino acid sequence of SEQ ID NO: 170in a light chain variable region having the amino acid sequence of SEQID NO: 52.

Substitution of Glu at position 5 of CDR3 (SEQ ID NO: 15) in the lightchain variable region of SEQ ID NO: 52 with Asp (SEQ ID NO: 193). Thus,the present invention provides light chain variable regions in whichCDR3 having the amino acid sequence of SEQ ID NO: 15 is substituted withCDR3 having the amino acid sequence of SEQ ID NO: 193 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Ser at position 6 of CDR3 (SEQ ID NO: 15) in the lightchain variable region of SEQ ID NO: 52 with Asp (SEQ ID NO: 194). Thus,the present invention provides light chain variable regions in whichCDR3 having the amino acid sequence of SEQ ID NO: 15 is substituted withCDR3 having the amino acid sequence of SEQ ID NO: 194 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

Substitution of Thr at position 9 of CDR3 (SEQ ID NO: 15) in the lightchain variable region of SEQ ID NO: 52 with Phe (SEQ ID NO: 195). Thus,the present invention provides light chain variable regions in whichCDR3 having the amino acid sequence of SEQ ID NO: 15 is substituted withCDR3 having the amino acid sequence of SEQ ID NO: 195 in a light chainvariable region having the amino acid sequence of SEQ ID NO: 52.

In addition, the substitutions other than those described above includea substitution of Arg at position 3 of heavy chain FR2 having the aminoacid sequence of SEQ ID NO: 97 with another amino acid. The amino acidafter substitution is not particularly limited; but preferred examplesthereof include Gln. When Arg at position 3 in SEQ ID NO: 97 has beenreplaced with Gln, Ala at position 5 may be substituted with Ser toproduce a human FR2 sequence. The amino acid sequence in which Arg andAla at positions 3 and 5 in the amino acid sequence of SEQ ID NO: 97have been replaced with Gln and Ser, respectively, is shown in SEQ IDNO: 120. Thus, the present invention provides heavy chain variableregions in which FR2 having the amino acid sequence of SEQ ID NO: 97 issubstituted with FR2 having the amino acid sequence of SEQ ID NO: 120 ina heavy chain variable region having the amino acid sequence of SEQ IDNO: 50 or 112.

Each of the above-described amino acid substitutions may be used aloneor in combination with other amino acid substitutions described above.They also may be combined with amino acid substitutions other than thosedescribed above.

Specific examples of the antibodies in which the above-describedsubstitutions have been carried out include, for example, antibodiesthat comprise a heavy chain variable region having the amino acidsequence of SEQ ID NO: 167, antibodies that comprise a light chainvariable region having the amino acid sequence of SEQ ID NO: 168, andantibodies that comprise a heavy chain variable region having the aminoacid sequence of SEQ ID NO: 167 and a light chain variable region havingthe amino acid sequence of SEQ ID NO: 168. Furthermore, specificexamples of the heavy chain variable regions in which theabove-described substitutions have been carried out include, forexample, the following heavy chain variable regions:

(1) heavy chain variable regions having the amino acid sequence of SEQID NO: 204 (H17);(2) heavy chain variable regions having the amino acid sequence of SEQID NO: 205 (H19);(3) heavy chain variable regions having the amino acid sequence of SEQID NO: 206 (H28);(4) heavy chain variable regions having the amino acid sequence of SEQID NO: 207 (H30);(5) heavy chain variable regions having the amino acid sequence of SEQID NO: 208 (H34);(6) heavy chain variable regions having the amino acid sequence of SEQID NO: 209 (H42);(7) heavy chain variable regions having the amino acid sequence of SEQID NO: 210 (H44);(8) heavy chain variable regions having the amino acid sequence of SEQID NO: 211 (H46);(9) heavy chain variable regions having the amino acid sequence of SEQID NO: 212 (H57);(10) heavy chain variable regions having the amino acid sequence of SEQID NO: 213 (H71);(11) heavy chain variable regions having the amino acid sequence of SEQID NO: 214 (H78);(12) heavy chain variable regions having the amino acid sequence of SEQID NO: 215 (H92);(13) heavy chain variable regions having the amino acid sequence of SEQID NO: 216 (H97); and(14) heavy chain variable regions having the amino acid sequence of SEQID NO: 217 (H98).

Meanwhile, specific examples of the light chain variable regions inwhich the above-described substitutions carried out include, forexample, the following light chain variable regions:

(15) light chain variable regions having the amino acid sequence of SEQID NO: 218 (L11);(16) light chain variable regions having the amino acid sequence of SEQID NO: 219 (L12);(17) light chain variable regions having the amino acid sequence of SEQID NO: 220 (L17); and(18) light chain variable regions having the amino acid sequence of SEQID NO: 221 (L50).

Furthermore, specific examples of the antibodies comprising theabove-described heavy chain and light chain variable regions include,for example, the following antibodies:

(19) antibodies that comprise the heavy chain variable region of (3) andthe light chain variable region of (17) (H28L17);(20) antibodies that comprise the heavy chain variable region of (4) andthe light chain variable region of (17) (H30L17);(21) antibodies that comprise the heavy chain variable region of (5) andthe light chain variable region of (17) (H34L17);(22) antibodies that comprise the heavy chain variable region of (6) andthe light chain variable region of (17) (H42L17);(23) antibodies that comprise the heavy chain variable region of (7) andthe light chain variable region of (17) (H44L17);(24) antibodies that comprise the heavy chain variable region of (8) andthe light chain variable region of (17) (H46L17);(25) antibodies that comprise the heavy chain variable region of (9) andthe light chain variable region of (17) (H57L17);(26) antibodies that comprise the heavy chain variable region of (10)and the light chain variable region of (17) (H71L17);(27) antibodies that comprise the heavy chain variable region of (11)and the light chain variable region of (17) (H78L17);(28) antibodies that comprise the heavy chain variable region of (12)and the light chain variable region of (17) (H92L17);(29) antibodies that comprise the heavy chain variable region of (13)and the light chain variable region of (18) (H97L50); and(30) antibodies that comprise the heavy chain variable region of (14)and the light chain variable region of (18) (H98L50).

The constant region used for the humanized antibodies of the presentinvention may be any constant region derived from a human antibody.Preferred examples of such constant regions derived from humanantibodies include, for example, constant regions derived from IgG1 orIgG2. Moreover, constant regions in which one or more amino acids aresubstituted, deleted, added, and/or inserted in the constant regionderived from a human antibody may also be used.

The constant regions in which one or more amino acids are substituted,deleted, added, and/or inserted in the constant region derived from ahuman antibody are not particularly limited, and include, for example,the following constant regions:

constant regions having the amino acid sequence of SEQ ID NO: 128 (M58);

constant regions having the amino acid sequence of SEQ ID NO: 129 (M14);and

constant regions having the amino acid sequence of SEQ ID NO: 62 (SKSC).

Specific examples of the heavy chains or antibodies having theabove-described constant regions include, for example:

(1) heavy chains that comprise a variable region having the amino acidsequence of SEQ ID NO: 167 and a constant region having the amino acidsequence of SEQ ID NO: 128;(2) heavy chains in which CDR2 having the amino acid sequence of SEQ IDNO: 171 in the heavy chains of (1) is substituted with CDR2 having theamino acid sequence of SEQ ID NO: 172;(3) antibodies that comprise the heavy chain of (1) and a light chainhaving the amino acid sequence of SEQ ID NO: 152; and(4) antibodies that comprise the heavy chain of (2) and a light chainhaving the amino acid sequence of SEQ ID NO: 152.

More specific examples of the humanized anti-NR10 antibodies of thepresent invention include, for example, the following antibodies:

(k) antibodies that comprise a heavy chain having the amino acidsequence of SEQ ID NO: 54 (H0-VH+constant region);(l) antibodies that comprise a heavy chain having the amino acidsequence of SEQ ID NO: 130 (H1-VH+constant region);(m) antibodies that comprise a light chain having the amino acidsequence of SEQ ID NO: 56 (L0-VL+constant region);(n) antibodies that comprise a heavy chain having the amino acidsequence of SEQ ID NO: 54 (H0-VH+constant region) and a light chainhaving the amino acid sequence of SEQ ID NO: 56 (L0-VL+constant region);and(o) antibodies that comprise a heavy chain having the amino acidsequence of SEQ ID NO: 130 (H1-VH+constant region) and a light chainhaving the amino acid sequence of SEQ ID NO: 56 (L0-VL+constant region).

The heavy chain having the amino acid sequence of SEQ ID NO: 54(H0-VH+constant region) and the light chain having the amino acidsequence of SEQ ID NO: 56 (L0-VL+constant region) may have asubstitution, deletion, addition, and/or insertion of one or more aminoacids. The substitution, deletion, addition, and/or insertion of aminoacids may be carried out in either or both of the variable and constantregions.

Thus, the present invention provides:

(p) antibodies that comprise a heavy chain having an amino acid sequencein which one or more amino acids are substituted, deleted, added, and/orinserted in the amino acid sequence of SEQ ID NO: 54 (H0-VH+constantregion);(q) antibodies that comprise a heavy chain having an amino acid sequencein which one or more amino acids are substituted, deleted, added, and/orinserted in the amino acid sequence of SEQ ID NO: 130 (H1-VH+constantregion);(r) antibodies that comprise a light chain having an amino acid sequencein which one or more amino acids are substituted, deleted, added, and/orinserted in the amino acid sequence of SEQ ID NO: 56 (L0-VL+constantregion);(s) antibodies that comprise a heavy chain having an amino acid sequencein which one or more amino acids are substituted, deleted, added, and/orinserted in the amino acid sequence of SEQ ID NO: 54 (H0-VH+constantregion) and a light chain having an amino acid sequence in which one ormore amino acids are substituted, deleted, added, and/or inserted in theamino acid sequence of SEQ ID NO: 56 (L0-VL+constant region); and(t) antibodies that comprise a heavy chain having an amino acid sequencein which one or more amino acids are substituted, deleted, added, and/orinserted in the amino acid sequence of SEQ ID NO: 130 (H1-VH+constantregion) and a light chain having an amino acid sequence in which one ormore amino acids are substituted, deleted, added, and/or inserted in theamino acid sequence of SEQ ID NO: 56 (L0-VL+constant region).

Without particular limitation, the antibodies of any one of (p) to (t)preferably have an activity similar to that of the antibodies of any oneof (k) to (o).

The substitution, deletion, addition, and/or insertion of amino acidsare not particularly limited, but specific examples thereof include, forexample, the above-described amino acid substitutions.

Furthermore, the nucleotide sequence encoding the amino acid sequence ofthe above-described humanized heavy chain variable region (SEQ ID NO:50) is shown in SEQ ID NO: 49. The nucleotide sequence encoding theamino acid sequence of the humanized light chain variable region (SEQ IDNO: 52) is shown in SEQ ID NO: 51. The nucleotide sequence encoding theamino acid sequence of the humanized heavy chain (SEQ ID NO: 54) isshown in SEQ ID NO: 53. The nucleotide sequence encoding the amino acidsequence of the humanized light chain (SEQ ID NO: 56) is shown in SEQ IDNO: 55.

Moreover, the present invention provides antibodies that recognize thesame epitope as recognized by the antibodies of any one of (a) to (t)above. The binding to the same epitope is as already described above.

Furthermore, the present invention provides the following antibodies:

(u) antibodies that comprise a heavy chain having the amino acidsequence of SEQ ID NO: 151;(v) antibodies that comprise a light chain comprising the amino acidsequence of SEQ ID NO: 152; and(w) antibodies that comprise the heavy chain of (u) and the light chainof (v). Moreover, the present invention provides the following heavy andlight chains and antibodies:(1) heavy chains having the amino acid sequence of SEQ ID NO: 222 (H17);(2) heavy chains having the amino acid sequence of SEQ ID NO: 223 (H19);(3) heavy chains having the amino acid sequence of SEQ ID NO: 224 (H28);(4) heavy chains having the amino acid sequence of SEQ ID NO: 225 (H30);(5) heavy chains having the amino acid sequence of SEQ ID NO: 226 (H34);(6) heavy chains having the amino acid sequence of SEQ ID NO: 227 (H42);(7) heavy chains having the amino acid sequence of SEQ ID NO: 228 (H44);(8) heavy chains having the amino acid sequence of SEQ ID NO: 229 (H46);(9) heavy chains having the amino acid sequence of SEQ ID NO: 230 (H57);(10) heavy chains having the amino acid sequence of SEQ ID NO: 231(H71);(11) heavy chains having the amino acid sequence of SEQ ID NO: 232(H78);(12) heavy chains having the amino acid sequence of SEQ ID NO: 233(H92);(13) heavy chains having the amino acid sequence of SEQ ID NO: 234(H97);(14) heavy chains having the amino acid sequence of SEQ ID NO: 235(H98);(15) light chains having the amino acid sequence of SEQ ID NO: 236 (L11)(16) light chains having the amino acid sequence of SEQ ID NO: 237(L12);(17) light chains having the amino acid sequence of SEQ ID NO: 238(L17);(18) light chains having the amino acid sequence of SEQ ID NO: 239(L50);(19) antibodies that comprise the heavy chain of (3) and the light chainof (17) (H28L17);(20) antibodies that comprise the heavy chain of (4) and the light chainof (17) (H30L17);(21) antibodies that comprise the heavy chain of (5) and the light chainof (17) (H34L17);(22) antibodies that comprise the heavy chain of (6) and the light chainof (17) (H42L17);(23) antibodies that comprise the heavy chain of (7) and the light chainof (17) (H44L17);(24) antibodies that comprise the heavy chain of (8) and the light chainof (17) (H46L17);(25) antibodies that comprise the heavy chain of (9) and the light chainof (17) (H57L17);(26) antibodies that comprise the heavy chain of (10) and the lightchain of (17) (H71L17);(27) antibodies that comprise the heavy chain of (11) and the lightchain of (17) (H78L17);(28) antibodies that comprise the heavy chain of (12) and the lightchain of (17) (H92L17);(29) antibodies that comprise the heavy chain of (13) and the lightchain of (18) (H97L50);(30) antibodies that comprise the heavy chain of (14) and the lightchain of (18) (H98L50);(31) heavy chains having an amino acid sequence in which one or moreamino acids are substituted, deleted, added and/or inserted in the heavychains of any one of (1) to (14);(32) light chains having an amino acid sequence in which one or moreamino acids are substituted, deleted, added and/or inserted in the lightchains of any one of (15) to (18);(33) antibodies having an amino acid sequence in which one or more aminoacids are substituted, deleted, added and/or inserted in the antibodiesof any one of (19) to (30); and(34) antibodies that recognize the same epitope as recognized by theantibodies of any one of (19) to (33).

The substitution, deletion, addition, and/or insertion of amino acidsare as described above. Antibodies that recognize the same epitope asrecognized by an antibody are also described above.

The present invention also provides genes encoding the variable regions,heavy chains, light chains, or antibodies of the present invention.

The present invention also provides vectors carrying the above-describedgenes.

The present invention also provides host cells transformed with theabove-described vectors.

The present invention also relates to methods for producing variableregions, heavy chains, light chains, or antibodies of the presentinvention, which comprise the step of culturing the above-described hostcells.

The vectors, host cells, and culture of host cells are described hereinbelow.

Antibodies that Recognize Domains

Preferred embodiments of the anti-NR10 antibody of the present inventioninclude antibodies that recognize domain 1 or domain 2. In the presentinvention, domain 1 refers to the region of amino acids at positions 21to 120 (LPAKP to LENIA) in the amino acid sequence of human NR10 of SEQID NO: 76, where the amino acid numbering is based on the sequenceincluding the signal peptide. In addition, in the present invention,domain 2 refers to the region of amino acids at positions 121 to 227(KTEPP to EEEAP) in the amino acid sequence of human NR10 of SEQ ID NO:76, where the amino acid numbering is based on the sequence includingthe signal peptide.

Such antibodies are not particularly limited; however, in general, theyhave a neutralizing activity, and preferably are humanized antibodies.

Examples of the preferred antibodies in the present invention includeantibodies that recognize domain 1. The antibodies that recognize domain1 have a strong neutralizing activity, and thus are particularly usefulas pharmaceuticals.

Antibodies (Neutralizing Activity)

The present invention also provides anti-NR10 antibodies having aneutralizing activity.

In the present invention, the neutralizing activity against NR10 refersto an activity of inhibiting the binding between NR10 and its ligandIL-31, and preferably an activity of suppressing a biological activitybased on NR10.

Antibodies having a NR10-neutralizing activity can be selected, forexample, by adding candidate antibodies to an IL-31-dependent cell lineand observing their growth-suppressing effect on the cell line. In thismethod, antibodies that suppress the growth of the IL-31-dependent cellline are determined as antibodies having a neutralizing activity againstNR10.

Antibodies (General)

The antibodies of the present invention are not limited in terms oftheir origin, and may be derived from any animals such as humans, mice,and rats. Moreover, the antibodies may be recombinant antibodies such aschimeric antibodies and humanized antibodies. As described above, thepreferred antibodies of the present invention include humanizedantibodies.

The chimeric antibodies contain, for example, the heavy and light chainconstant regions of a human antibody, and the heavy and light chainvariable regions of an antibody of a non-human mammal, such as mouse.The chimeric antibodies can be produced by known methods. For example,the antibodies can be produced by cloning an antibody gene fromhybridomas, inserting it into an appropriate vector, and introducing theconstruct into hosts (see, for example, Carl, A. K. Borrebaeck, James,W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the UnitedKingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNAs of theantibody variable regions (V regions) are synthesized from mRNA ofhybridomas using reverse transcriptase. Once DNAs encoding the V regionsof an antibody of interest are obtained, these are linked with DNAsencoding the constant regions (C regions) of a desired human antibody.The resulting constructs are inserted into expression vectors.Alternatively, the DNAs encoding the antibody V regions may be insertedinto expression vectors comprising DNAs encoding the C regions of ahuman antibody. The DNAs are inserted into expression vectors so thatthey are expressed under the regulation of the expression regulatoryregions, for example, enhancers and promoters. In the next step, hostcells can be transformed with the expression vectors to allow expressionof chimeric antibodies.

Methods for obtaining human antibodies are also known. For example,desired human antibodies with antigen-binding activity can be obtainedby (1) sensitizing human lymphocytes with antigens of interest or cellsexpressing antigens of interest in vitro; and (2) fusing the sensitizedlymphocytes with human myeloma cells such as U266 (see Japanese PatentApplication Kokoku Publication No. (JP-B) H01-59878 (examined, approvedJapanese patent application published for opposition)). Alternatively,the desired human antibody can also be obtained by immunizing atransgenic animal having an entire repertoire of human antibody geneswith a desired antigen (see International Patent Application PublicationNos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096,and WO 96/33735).

Furthermore, techniques to obtain human antibodies by panning with ahuman antibody phage library are known. For example, the variable regionof a human antibody is expressed as a single chain antibody (scFv) onthe surface of a phage, using a phage display method, and phages thatbind to the antigen can be selected. By analyzing the genes of selectedphages, the DNA sequences encoding the variable regions of humanantibodies that bind to the antigen can be determined. If the DNAsequences of scFvs that bind to the antigen are identified, appropriateexpression vectors comprising these sequences can be constructed toobtain human antibodies. Such methods are well known. Reference can bemade to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172,WO 95/01438, WO 95/15388, and such.

The antibodies of the present invention include not only divalentantibodies as represented by IgG, but also monovalent antibodies,multivalent antibodies as represented by IgM, and bispecific antibodiescapable of binding to different antigens, as long as they have aNR10-binding activity and/or neutralizing activity. The multivalentantibodies of the present invention include multivalent antibodies inwhich the antigen-binding sites are all identical, and multivalentantibodies in which all or some of the antigen-binding sites aredifferent. The antibodies of the present invention are not limited tofull-length antibody molecules, but may also be low-molecular-weightantibodies or modified products thereof, as long as they bind to NR10protein.

Alternatively, the antibodies of the present invention may below-molecular-weight antibodies. Such low-molecular-weight antibodiesare antibodies including antibody fragments lacking some portions of awhole antibody (for example, whole IgG), and are not particularlylimited as long as they retain NR10-binding and/or neutralizingactivity. In the present invention, the low-molecular-weight antibodiesare not particularly limited, as long as they contain a portion of wholeantibodies. The low-molecular-weight antibodies preferably contain aheavy chain variable region (VH) or light chain variable region (VL).Particularly preferred low-molecular-weight antibodies contain both VHand VL. In addition, preferred examples of the low-molecular-weightantibodies of the present invention include low-molecular-weightantibodies containing CDRs of an antibody. The CDRs contained in thelow-molecular-weight antibodies may include some or all of the six CDRsof an antibody.

The low-molecular-weight antibodies of the present invention preferablyhave a smaller molecular weight than whole antibodies. However, thelow-molecular-weight antibodies may form multimers, for example, dimers,trimers, or tetramers, and thus their molecular weights can be greaterthan those of whole antibodies.

Specific examples of the antibody fragments include, for example, Fab,Fab′, F(ab′)2, and Fv. Meanwhile, specific examples of thelow-molecular-weight antibodies include, for example, Fab, Fab′,F(ab′)2, Fv, scFv (single chain Fv), diabodies, and sc(Fv)2 (singlechain (Fv)2). Multimers (for example, dimers, trimers, tetramers, andpolymers) of these antibodies are also included in thelow-molecular-weight antibodies of the present invention.

Antibody fragments can be obtained, for example, by treating antibodieswith enzymes to produce antibody fragments. Enzymes known to generateantibody fragments include, for example, papain, pepsin, and plasmin.Alternatively, a gene encoding such an antibody fragment can beconstructed, introduced into an expression vector, and expressed inappropriate host cells (see, for example, Co, M. S. et al., J. Immunol.(1994)152, 2968-2976; Better, M. & Horwitz, A. H. Methods in Enzymology(1989)178, 476-496; Plueckthun, A. & Skerra, A. Methods in Enzymology(1989)178, 476-496; Lamoyi, E., Methods in Enzymology (1989)121,652-663; Rousseaux, J. et al., Methods in Enzymology (1989)121, 663-669;Bird, R. E. et al., TIBTECH (1991)9, 132-137).

Digestive enzymes cleave a specific site of an antibody fragment,yielding antibody fragments of specific structures shown below. Geneticengineering techniques can be applied to such enzymatically-obtainedantibody fragments to delete an arbitrary portion of the antibody.

Antibody fragments obtained by using the above-described digestiveenzymes are as follows:

Papain digestion: F(ab)2 or FabPepsin digestion: F(ab′)2 or Fab′Plasmin digestion: Facb

The low-molecular-weight antibodies of the present invention includeantibody fragments lacking an arbitrary region, as long as they have aNR10-binding activity and/or neutralizing activity.

“Diabody” refers to a bivalent antibody fragment constructed by genefusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993); EP 404,097; WO 93/11161, etc). Diabodies are dimers composed oftwo polypeptide chains. In each of the polypeptide chains forming adimer, a VL and a VH are usually linked by a linker in the same chain.In general, the linker in a diabody is short enough such that the VL andVH cannot bind to each other. Specifically, the number of amino acidresidues constituting the linker is, for example, about five residues.Thus, the VL and VH encoded on the same polypeptide cannot form asingle-chain variable region fragment, and will form a dimer withanother single-chain variable region fragment. As a result, the diabodyhas two antigen binding sites.

ScFv antibodies are single-chain polypeptides produced by linking aheavy chain variable region ([VH]) and a light chain variable region([VL]) via a linker or such (Huston, J. S. et al., Proc. Natl. Acad.Sci. U.S.A. (1988) 85, 5879-5883; Pluckthun “The Pharmacology ofMonoclonal Antibodies” Vol. 113, eds., Resenburg and Moore, SpringerVerlag, New York, pp. 269-315, (1994)). The H-chain V region and L-chainV region of scFv may be derived from any antibody described herein. Thepeptide linker for linking the V regions is not particularly limited.For example, an arbitrary single-chain peptide containing about three to25 residues can be used as the linker. Specifically, it is possible touse the peptide linkers or such described below.

The V regions of both chains can be linked, for example, by PCR asdescribed above. First, among the following DNAs, a DNA encoding acomplete or desired partial amino acid sequence is used as a template tolink the V regions by PCR:

DNA sequence encoding an H chain or H-chain V region of an antibody, and

DNA sequence encoding an L chain or L-chain V region of an antibody.

DNAs encoding the V regions of H chain and L chain are amplified by PCRusing a pair of primers having sequences corresponding to those at bothends of the DNA to be amplified. Then, a DNA encoding the peptide linkerportion is prepared. The peptide linker-encoding DNA can also besynthesized by PCR. Here, nucleotide sequences that can be ligated tothe amplification products of V regions synthesized separately are addedto the 5′ end of the primers to be used. Then, PCR is carried out usingeach DNA of the [H chain V region DNA]-[peptide linker DNA]-[L chain Vregion DNA], and assembly PCR primers.

The assembly PCR primers are composed of a combination of a primer thatanneals to the 5′ end of the [H chain V region DNA] and a primer thatanneals to the 3′ end of the [L chain V region DNA]. In other words, theassembly PCR primers are a set of primers that can be used to amplifyDNA encoding the full-length sequence of scFv to be synthesized.Meanwhile, nucleotide sequences that can be ligated to the V-region DNAshave been added to the [peptide linker DNA]. Thus, these DNAs are linkedtogether, and then the whole scFv is ultimately generated as anamplification product by the assembly PCR primers. Once thescFv-encoding DNAs are generated, expression vectors carrying these DNAsand recombinant cells transformed with these expression vectors can beobtained by conventional methods. Furthermore, the scFv can be obtainedby culturing the resulting recombinant cells to express thescFv-encoding DNAs.

The order of the heavy chain and light chain variable regions to belinked together is not particularly limited, and they may be arranged inany order. Examples of the arrangement are listed below.

[VH] linker [VL]

[VL] linker [VH]

sc(Fv)2 is a single-chain low-molecular-weight antibody produced bylinking two VHs and two VLs using linkers and such (Hudson et al., JImmunol. Methods 1999; 231: 177-189). For example, sc(Fv)2 can beproduced by linking scFvs via a linker.

Antibodies in which two VHs and two VLs are arranged in the order ofVH-VL-VH-VL ([VH] linker [VL] linker [VH] linker [VL]) from the Nterminus of the single-chain polypeptide are preferred. However, theorder of the two VHs and two VLs is not limited to the abovearrangement, and they may be arranged in any order. Examples of thearrangement are listed below:

[VL] linker [VH] linker [VH] linker [VL]

[VH] linker [VL] linker [VL] linker [VH]

[VH] linker [VH] linker [VL] linker [VL]

[VL] linker [VL] linker [VH] linker [VH]

[VL] linker [VH] linker [VL] linker [VH]

The amino acid sequence of the heavy chain variable region or lightchain variable region in a low-molecular-weight antibody may contain asubstitution, deletion, addition, and/or insertion. Furthermore, theheavy chain variable region and light chain variable region may alsolack some portions or be added with other polypeptides, as long as theyhave antigen binding ability when linked together. Alternatively, thevariable regions may be chimerized or humanized.

In the present invention, linkers which bind the variable regions of theantibody include arbitrary peptide linkers that can be introduced usinggenetic engineering, or synthetic linkers such as those disclosed inProtein Engineering, 9(3), 299-305, 1996.

The preferred linkers in the present invention are peptide linkers. Thelengths of the peptide linkers are not particularly limited and thoseskilled in the art can appropriately select the lengths depending on thepurpose. Typical lengths are one to 100 amino acids, preferably 3 to 50amino acids, more preferably 5 to 30 amino acids, and particularlypreferably 12 to 18 amino acids (for example, 15 amino acids).

Amino acid sequences of such peptide linkers include, for example:

Ser; Gly-Ser; Gly-Gly-Ser; Ser-Gly-Gly; (SEQ ID NO: 82) Gly-Gly-Gly-Ser;(SEQ ID NO: 83) Ser-Gly-Gly-Gly; (SEQ ID NO: 84) Gly-Gly-Gly-Gly-Ser;(SEQ ID NO: 85) Ser-Gly-Gly-Gly-Gly; (SEQ ID NO: 86)Gly-Gly-Gly-Gly-Gly-Ser; (SEQ ID NO: 87) Ser-Gly-Gly-Gly-Gly-Gly;(SEQ ID NO: 88) Gly-Gly-Gly-Gly-Gly-Gly-Ser; (SEQ ID NO: 89)Ser-Gly-Gly-Gly-Gly-Gly-Gly; (SEQ ID NO: 84) (Gly-Gly-Gly-Gly-Ser )n;and (SEQ ID NO: 85) (Ser-Gly-Gly-Gly-Gly )n,where n is an integer of 1 or larger.

The amino acid sequence of peptide linker can be appropriately selectedby those skilled in the art depending on the purpose. For example, theabove-mentioned “n”, which determines the length of the peptide linker,is usually 1 to 5, preferably 1 to 3, and more preferably 1 or 2.

Synthetic linkers (chemical crosslinking agents) include crosslinkingagents that are routinely used to crosslink peptides, for example,N-hydroxy succinimide (NHS), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS³), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP),ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycolbis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate(DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidoxycarbonyloxy)ethyl] sulfone (BSOCOES), andbis[2-(sulfosuccinimidoxycarbonyloxy)ethyl] sulfone (sulfo-BSOCOES).These crosslinking agents are commercially available.

When four antibody variable regions are linked, three linkers areusually required. Such multiple linkers may be the same or different.

The antibodies of the present invention include antibodies in which oneor more amino acid residues have been added to the amino acid sequenceof an antibody of the present invention. Further, fusion proteins whichresult from a fusion between one of the above antibodies and a secondpeptide or protein is included in the present invention. The fusionproteins can be prepared by ligating a polynucleotide encoding anantibody of the present invention and a polynucleotide encoding a secondpeptide or polypeptide in frame, inserting this into an expressionvector, and expressing the fusion construct in a host. Some techniquesknown to those skilled in the art are available for this purpose. Thepartner peptide or polypeptide to be fused with an antibody of thepresent invention may be a known peptide, for example, FLAG (Hopp, T. P.et al., BioTechnology 6, 1204-1210 (1988)), 6× His consisting of six His(histidine) residues, 10× His, influenza hemagglutinin (HA), human c-mycfragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein Cfragment. Other partner polypeptides to be fused with the antibodies ofthe present invention include, for example, GST(glutathione-S-transferase), HA (influenza hemagglutinin),immunoglobulin constant region, β-galactosidase, and MBP(maltose-binding protein). A polynucleotide encoding one of thesecommercially available peptides or polypeptides can be fused with apolynucleotide encoding an antibody of the present invention. The fusionpolypeptide can be prepared by expressing the fusion construct.

Furthermore, the antibodies of the present invention may be conjugatedantibodies which are linked to any of various molecules includingpolymeric substances such as polyethylene glycol (PEG) and hyaluronicacid, radioactive substances, fluorescent substances, luminescentsubstances, enzymes, and toxins. Such conjugated antibodies can beobtained by chemically modifying the obtained antibodies. Methods formodifying antibodies have been established in this field (for example,U.S. Pat. Nos. 5,057,313 and 5,156,840). The “antibodies” of the presentinvention also include such conjugated antibodies.

Furthermore, the antibodies used in the present invention may bebispecific antibodies. The bispecific antibody refers to an antibodythat has variable regions recognizing different epitopes in the sameantibody molecule. In the present invention, the bispecific antibodiesmay recognize different epitopes on an NR10 molecule, or recognize NR10with one antigen-binding site and a different substance with the otherantigen-binding site.

Methods for producing bispecific antibodies are known. Bispecificantibodies can be prepared, for example, by linking two antibodies thatrecognize different antigens. Antibodies to be linked together may behalf molecules each of which contains an H chain and an L chain, orquarter molecules that consist of only one H chain. Alternatively,hybridomas producing different monoclonal antibodies can be fused toproduce a bispecific antibody-producing fused cell. Furthermore,bispecific antibodies can be produced by genetic engineering techniques.

The antibodies of the present invention may differ in amino acidsequence, molecular weight, isoelectric point, presence/absence of sugarchains, and conformation depending on the cell or host producing theantibody or the purification method as described below. However, aresulting antibody is included in the present invention, as long as itis functionally equivalent to an antibody of the present invention. Forexample, when an antibody of the present invention is expressed inprokaryotic cells, for example E. coli, a methionine residue is added tothe N terminus of the original antibody amino acid sequence. Suchantibodies are included in the present invention.

Antibody Production

The antibodies of the present invention may be polyclonal or monoclonalantibodies. Such monoclonal antibodies having NR10-binding and/orneutralizing activity can be obtained, for example, by the followingprocedure: anti-NR10 monoclonal antibodies are prepared by using as anantigen NR10 or a fragment thereof that is derived from a mammal such ashuman or mouse by known methods, and then antibodies having NR10-bindingand/or neutralizing activity are selected from the thus obtainedanti-NR10 monoclonal antibodies. Specifically, a desired antigen orcells expressing the desired antigen are used as a sensitizing antigenfor immunization according to conventional immunization methods.Anti-NR10 monoclonal antibodies can be prepared by fusing the obtainedimmune cells with known parental cells using conventional cell fusionmethods, and screening them for monoclonal antibody-producing cells(hybridomas) by conventional screening methods. Animals to be immunizedinclude, for example, mammals such as mice, rats, rabbits, sheep,monkeys, goats, donkeys, cows, horses, and pigs. The antigen can beprepared using the known NR10 gene sequence according to known methods,for example, by methods using baculovirus (for example, WO 98/46777).

Hybridomas can be prepared, for example, according to the method ofMilstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981)73: 3-46) or such. When the immunogenicity of an antigen is low,immunization may be performed after linking the antigen with amacromolecule having immunogenicity, such as albumin.

Embodiments of the antibodies of the present invention that have abinding and/or neutralizing activity against NR10 include monoclonalantibodies that have a binding and/or neutralizing activity againsthuman NR10. Antigens used to prepare monoclonal antibodies that have abinding and/or neutralizing activity against human NR10 are notparticularly limited, as long as they enable preparation of antibodiesthat have a binding and/or neutralizing activity against human NR10. Forexample, it is known that there are a number of variants of human NR10,and any variant may be used as an immunogen as long as it enablespreparation of antibodies that have a binding and/or neutralizingactivity against human NR10. Alternatively, under the same condition, apeptide fragment of NR10 or a protein in which artificial mutations havebeen introduced into the natural NR10 sequence may be used as animmunogen. Human NR10.3 is one of preferred immunogens in preparingantibodies that have an activity of binding and/or neutralizing NR10 inthe present invention.

Furthermore, the binding and/or neutralizing activity of antibodyagainst NR10 can be measured, for example, by observing the effect ofsuppressing the growth of the IL-31-dependent cell line as described inthe Examples.

Meanwhile, monoclonal antibodies can also be obtained by DNAimmunization. DNA immunization is a method in which a vector DNAconstructed such that the gene encoding an antigen protein can beexpressed in an animal to be immunized is administered to the animal,and the immunogen is expressed within the body of the animal to provideimmunostimulation. As compared to common immunization methods based onthe administration of protein antigens, the DNA immunization is expectedto be advantageous in that:

it enables immunostimulation while retaining the structure of a membraneprotein; and

the immunogen does not need to be purified.

On the other hand, it is difficult to combine DNA immunization with animmunostimulating means such as an adjuvant.

In order to obtain a monoclonal antibody by DNA immunization, first, DNAencoding NR10 is administered to an animal to be immunized. The DNAencoding NR10 can be synthesized by known methods such as PCR. Theresulting DNA is inserted into an appropriate expression vector, andadministered to the animal to be immunized. Expression vectors that canbe used include commercially available expression vectors such aspcDNA3.1. The vector can be administered to the living body byconventional methods. For example, DNA immunization can be carried outby introducing gold particles coated with the expression vector intocells by gene gun. Booster using NR10-expressing cells after DNAimmunization is a preferred method to yield a monoclonal antibody.

Once the mammal is immunized as described above and the serum level of adesired antibody is confirmed to be increased, immune cells arecollected from the mammal and subjected to cell fusion. Preferred immunecells are spleen cells in particular.

Mammalian myeloma cells are used for fusion with the above immune cells.It is preferred that myeloma cells have appropriate selection markersfor screening. The selection marker refers to a phenotype that allows(or does not allow) survival under particular culture conditions. Knownselection markers include hypoxanthine-guanine phosphoribosyltransferasedeficiency (hereinafter abbreviated as “HGPRT deficiency”) and thymidinekinase deficiency (hereinafter abbreviated as “TK deficiency”). HGPRT-or TK-deficient cells exhibit hypoxanthine-aminopterin-thymidinesensitivity (hereinafter abbreviated as “HAT sensitivity”). In HATselection medium, HAT-sensitive cells cannot synthesize DNA and thuswill die. However, when fused with normal cells, they can continue tosynthesize DNA via the salvage pathway of the normal cells and thus cangrow even in HAT selection medium.

HGPRT- or TK-deficient cells can be selected using a medium containing6-thioguanine, 8-azaguanine (hereinafter abbreviated as “8AG”), or5′-bromodeoxyuridine. While normal cells are killed due to incorporationof these pyrimidine analogs into DNA, cells lacking these enzymes cansurvive in the selection medium because they cannot incorporate thesepyrimidine analogs. Another selection marker called G418 resistanceconfers resistance to 2-deoxystreptamine antibiotics (gentamicinanalogs) due to the neomycin resistance gene. Various myeloma cellssuitable for cell fusion are known.

Cell fusion between immune cells and myeloma cells can be essentiallycarried out according to known methods, for example, the method byKohler and Milstein (Kohler. G. and Milstein, C., Methods Enzymol.(1981) 73, 3-46).

More specifically, cell fusion can be carried out, for example, in acommon culture medium in the presence of a cell fusion-promoting agent.The fusion-promoting agent includes, for example, polyethylene glycol(PEG) and Sendai virus (HVJ). If required, an auxiliary agent such asdimethyl sulfoxide may also be added to improve fusion efficiency.

The immune cells and myeloma cells may be used at an arbitrarilydetermined ratio. For example, the ratio of immune cells to myelomacells is preferably from 1 to 10. Culture media to be used for cellfusion include, for example, media that are suitable for the cell growthof myeloma cell line, such as RPMI 1640 and MEM, and other commonculture media used for this type of cell culture. In addition, theculture media may also be supplemented with serum supplement such asfetal calf serum (FCS).

Predetermined amounts of immune cells and myeloma cells are mixed wellin the culture medium, and then mixed with a PEG solution pre-heated to37° C. to produce fused cells (hybridomas). In the cell fusion method,for example, PEG with mean molecular weight of about 1,000-6,000 can beadded to the cells typically at a concentration of 30% to 60% (w/v).Then, successive addition of the appropriate culture medium listed aboveand removal of supernatant by centrifugation are repeated to eliminatethe cell fusion agent and such, which are unfavorable to the growth ofhybridomas.

The resulting hybridomas can be screened using a selection mediumaccording to the selection marker possessed by myeloma cells used in thecell fusion. For example, HGPRT- or TK-deficient cells can be screenedby culturing them in a HAT medium (a medium containing hypoxanthine,aminopterin, and thymidine). Specifically, when HAT-sensitive myelomacells are used in cell fusion, cells successfully fused with normalcells can be selectively grown in the HAT medium. The cell culture usingthe above HAT medium is continued for a sufficient period of time toallow all cells except the desired hybridomas (non-fused cells) to die.Specifically, in general, the desired hybridomas can be selected byculturing the cells for several days to several weeks. Then, screeningand single cloning of hybridomas that produce an antibody of interestcan be carried out by performing ordinary limiting dilution methods.Alternatively, antibodies that recognize NR10 can be prepared by themethod described in WO 03/104453.

Screening and single cloning of an antibody of interest can be suitablycarried out by known screening methods based on antigen-antibodyreaction. For example, an antigen is bound to a carrier such as beadsmade of polystyrene or such and commercially available 96-wellmicrotiter plates, and then reacted with the culture supernatant ofhybridoma. Next, the carrier is washed and then reacted with anenzyme-labeled secondary antibody or such. When the culture supernatantcontains an antibody of interest reactive to the sensitizing antigen,the secondary antibody binds to the carrier via this antibody. Finally,the secondary antibody bound to the carrier is detected to determinewhether the culture supernatant contains the antibody of interest.Hybridomas producing a desired antibody capable of binding to theantigen can be cloned by the limiting dilution method or such. Not onlythe antigen used for immunization but also an NR10 protein substantiallyequivalent thereto can be preferably used as an antigen for thispurpose. For example, a cell line expressing NR10, the extracellulardomain of NR10, or an oligopeptide composed of a partial amino acidsequence constituting the domain may be used as the antigen.

In addition to the above-described method for preparing hybridomasthrough immunization of a nonhuman animal with an antigen, antibodies ofinterest can also be obtained by sensitizing human lymphocytes with anantigen. Specifically, first, human lymphocytes are sensitized with anNR10 protein in vitro. Then, the sensitized lymphocytes are fused withan appropriate fusion partner. For example, human-derived myeloma cellswith the ability to divide permanently can be used as the fusion partner(see Japanese Patent Application Kokoku Publication No. (JP-B) H1-59878(examined, approved Japanese patent application published foropposition). Antibodies obtained by this method are human antibodieshaving an activity of binding to the NR10 protein.

The nucleotide sequence encoding an anti-NR10 antibody obtained by theabove-described method or such, and its amino acid sequence can beobtained by methods known to those skilled in the art.

Based on the obtained sequence of the anti-NR10 antibody, the anti-NR10antibody can be prepared, for example, by genetic recombinationtechniques known to those skilled in the art. Specifically, apolynucleotide encoding an antibody can be constructed based on thesequence of the NR10-recognizing antibody, inserted into an expressionvector, and then expressed in appropriate host cells (see for example,Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. andHorwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. andSkerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., MethodsEnzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol.(1986) 121, 663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol.(1991) 9, 132-137).

The vectors include M13 vectors, pUC vectors, pBR322, pBluescript, andpCR-Script. Alternatively, when aiming to subclone and excise cDNA, thevectors include, for example, pGEM-T, pDIRECT, and pT7, in addition tothe vectors described above. Expression vectors are particularly usefulwhen using vectors for producing the antibodies of the presentinvention. For example, when aiming for expression in E. coli such asJM109, DH5α, HB101, and XL1-Blue, the expression vectors not only havethe above-described characteristics that allow vector amplification inE. coli, but must also carry a promoter that allows efficient expressionin E. coli, for example, lacZ promoter (Ward et al., Nature (1989) 341,544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al.,Science (1988) 240, 1041-1043), T7 promoter or such. Such vectorsinclude pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, orpET (in this case, the host is preferably BL21 that expresses T7 RNApolymerase) in addition to the vectors described above.

The vectors may contain signal sequences for antibody secretion. As asignal sequence for antibody secretion, a pelB signal sequence (Lei, S.P. et al J. Bacteriol. (1987) 169, 4379) may be used when a protein issecreted into the E. coli periplasm. The vector can be introduced intohost cells by calcium chloride or electroporation methods, for example.

In addition to vectors for E. coli, the vectors for producing theantibodies of the present invention include mammalian expression vectors(for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. 1990,18(17), p5322), pEF, and pCDM8), insect cell-derived expression vectors(for example, the “Bac-to-BAC baculovirus expression system” (Gibco-BRL)and pBacPAK8), plant-derived expression vectors (for example, pMH1 andpMH2), animal virus-derived expression vectors (for example, pHSV, pMV,and pAdexLcw), retroviral expression vectors (for example, pZIPneo),yeast expression vectors (for example, “Pichia Expression Kit”(Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expressionvectors (for example, pPL608 and pKTH50), for example.

When aiming for expression in animal cells such as CHO, COS, and NIH3T3cells, the vectors must have a promoter essential for expression incells, for example, SV40 promoter (Mulligan et al., Nature (1979) 277,108), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic AcidsRes. (1990) 18, 5322), and CMV promoter, and more preferably they have agene for selecting transformed cells (for example, a drug resistancegene that allows evaluation using an agent (neomycin, G418, or such).Vectors with such characteristics include pMAM, pDR2, pBK-RSV, pBK-CMV,pOPRSV, and pOP13, for example.

In addition, the following method can be used for stable gene expressionand gene amplification in cells: CHO cells deficient in a nucleic acidsynthesis pathway are introduced with a vector (for example, pSV2-dhfr(Molecular Cloning 2^(nd) edition, Cold Spring Harbor Laboratory Press,1989)) that carries a DHFR gene which compensates for the deficiency,and the vector is amplified using methotrexate (MTX). Alternatively, thefollowing method can be used for transient gene expression: COS cellswith a gene expressing SV40 T antigen on their chromosome aretransformed with a vector (pcD and such) with an SV40 replicationorigin. Replication origins derived from polyoma virus, adenovirus,bovine papilloma virus (BPV), and such can also be used. To amplify genecopy number in host cells, the expression vectors may further carryselection markers such as aminoglycoside transferase (APH) gene,thymidine kinase (TK) gene, E. coli xanthine-guaninephosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase(dhfr) gene.

The antibodies of the present invention obtained by the methodsdescribed above can be isolated from inside host cells or from outsidethe cells (the medium, or such), and purified to homogeneity. Theantibodies can be isolated and purified by methods routinely used forisolating and purifying antibodies, and the type of method is notlimited. For example, the antibodies can be isolated and purified byappropriately selecting and combining column chromatography, filtration,ultrafiltration, salting out, solvent precipitation, solvent extraction,distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectrofocusing, dialysis, recrystallization, andsuch.

The chromatographies include, for example, affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtration,reverse phase chromatography, and adsorption chromatography (Strategiesfor Protein Purification and Characterization: A Laboratory CourseManual. Ed Daniel R. Marshak et al., Cold Spring Harbor LaboratoryPress, 1996). The chromatographic methods described above can beconducted using liquid chromatography, for example, HPLC and FPLC.Columns that can be used for affinity chromatography include protein Acolumns and protein G columns. Columns using protein A include, forexample, Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). Thepresent invention includes antibodies that are highly purified usingthese purification methods.

The NR10-binding activity of the obtained antibodies can be determinedby methods known to those skilled in the art. Methods for determiningthe antigen-binding activity of an antibody include, for example, ELISA(enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA(radioimmunoassay), and fluorescent antibody method. For example, whenenzyme immunoassay is used, antibody-containing samples, such aspurified antibodies and culture supernatants of antibody-producingcells, are added to antigen-coated plates. A secondary antibody labeledwith an enzyme, such as alkaline phosphatase, is added and the platesare incubated. After washing, an enzyme substrate, such as p-nitrophenylphosphate, is added, and the absorbance is measured to evaluate theantigen-binding activity.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositionscomprising the antibody mentioned above as an active ingredient.Moreover, the present invention provides therapeutic agents forinflammatory diseases which comprise the antibody mentioned above as anactive ingredient.

In the present invention, inflammatory disease refers to diseases withpathological features involved in cytological and histological reactionsthat occur in affected blood vessels and adjacent tissues in response toan injury or abnormal stimulation caused by physical, chemical, orbiological agents (Stedman's Medical Dictionary, 5th Ed., MEDICAL VIEWCO., 2005). Generally, inflammatory diseases include, dermatitis (atopicdermatitis, chronic dermatitis, and such), inflammatory bowel diseases(colitis and such), asthma, arthritis (rheumatoid arthritis,osteoarthritis, and such), bronchitis, Th2 autoimmune diseases, systemiclupus erythematosus, myasthenia gravis, chronic GVHD, Crohn's disease,spondylitis deformans, lumbar pain, gout, inflammation after surgery orinjury, swelling, neuralgia, laryngopharyngitis, cystitis, hepatitis(non-alcoholic steatohepatitis, alcoholic hepatitis, and such),hepatitis B, hepatitis C, arteriosclerosis, and pruritus.

Preferred examples of inflammatory diseases that are subjects of thepresent invention include atopic dermatitis, chronic dermatitis,rheumatism, osteoarthritis, chronic asthma, and pruritus.

The phrase “comprise(s) an anti-NR10 antibody as an active ingredient”means comprising an anti-NR10 antibody as at least one of the activeingredients, and does not limit the proportion of the antibody. Inaddition, the therapeutic agents for inflammatory diseases in thepresent invention may also comprise, in combination with the anti-NR10antibody mentioned above, other ingredients that enhance the treatmentof inflammatory diseases.

The therapeutic agents of the present invention may also be used forpreventive purposes.

The anti-NR10 antibody of the present invention may be prepared asformulations according to standard methods (see, for example,Remington's Pharmaceutical Science, latest edition, Mark PublishingCompany, Easton, USA). Further, they may contain pharmaceuticallyacceptable carriers and/or additives if necessary. For example, they maycontain surfactants (for example, PEG and Tween), excipients,antioxidants (for example, ascorbic acid), coloring agents, flavoringagents, preservatives, stabilizers, buffering agents (for example,phosphoric acid, citric acid, and other organic acids), chelating agents(for example, EDTA), suspending agents, isotonizing agents, binders,disintegrators, lubricants, fluidity promoters, and corrigents. However,without limitation to these, the agents for preventing or treatinginflammatory diseases of the present invention may contain othercommonly used carriers. Such carriers specifically include lightanhydrous silicic acid, lactose, crystalline cellulose, mannitol,starch, carmelose calcium, carmelose sodium, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylacetaldiethylaminoacetate,polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, sucrose,carboxymethylcellulose, corn starch, and inorganic salt. The agents mayalso contain other low-molecular-weight polypeptides, proteins such asserum albumin, gelatin, and immunoglobulin, and amino acids such asglycine, glutamine, asparagine, arginine, and lysine. When the anti-NR10antibody is prepared as an aqueous solution for injection, the anti-NR10antibody may be dissolved in an isotonic solution containing, forexample, physiological saline, dextrose, or other adjuvants. Theadjuvants may include, for example, D-sorbitol, D-mannose, D-mannitol,and sodium chloride. In addition, appropriate solubilizing agents, forexample, alcohols (for example, ethanol), polyalcohols (for example,propylene glycols and PEGs), and non-ionic detergents (polysorbate 80and HCO-50) may be used concomitantly.

If necessary, anti-NR10 antibodies may be encapsulated in microcapsules(microcapsules made of hydroxymethylcellulose, gelatin,polymethylmethacrylate, and the like), and made into components ofcolloidal drug delivery systems (liposomes, albumin microspheres,microemulsions, nano-particles, and nano-capsules) (for example, see“Remington's Pharmaceutical Science 16th edition” &, Oslo Ed. (1980)).Moreover, methods for making sustained-release drugs are known, andthese can be applied for anti-NR10 antibodies (Langer et al., J. Biomed.Mater. Res. (1981) 15, 167-277; Langer, Chem. Tech. (1982) 12, 98-105;U.S. Pat. No. 3,773,919; European Patent Application (EP) No. 58,481;Sidman et al., Biopolymers (1983) 22, 547-56; EP 133,988).

The pharmaceutical compositions of the present invention can beadministered either orally or parenterally, but are preferablyadministered parenterally. Specifically, the agents are administered topatients by injection or percutaneous administration. Injectionsinclude, for example, intravenous injections, intramuscular injections,and subcutaneous injections, for systemic or local administration. Theagents may be given to sites where inflammation is to be suppressed, orareas surrounding the sites by local infusion, intramuscular injectionin particular. The administration methods can be properly selectedaccording to the patient's age and condition. The single-administrationdose can be selected, for example, from within the range of 0.0001 to100 mg of the active ingredient per kg body weight. Alternatively, forexample, when the agents are administered to human patients, the dose ofthe active ingredient can be selected from within the range of 0.001 to1,000 mg/kg body weight. The single-administration dose preferablycontains, for example, about 0.01 to 50 mg/kg body weight of theantibody of the present invention. However, the dose of an agent forpreventing or treating inflammatory diseases of the present invention isnot limited to these examples.

All prior-art documents cited in the present specification are hereinincorporated by reference.

EXAMPLES

Herein below, the present invention will be specifically described withreference to Examples, but it is not to be construed as being limitedthereto.

[Example 1] Preparation of Hybridomas 1.1. Preparation of Human andCynomolgus Monkey NR10 Plasmids for DNA Immunization

1.1.1. Preparation of Expression Vectors for hNR10 and cynNR10

Human NR10 (nucleotide sequence, SEQ ID NO: 75; amino acid sequence, SEQID NO: 76) was inserted into the expression vector pMacII, whichexpresses a protein under the control of mouse β-actin promoter(WO2005/054467), to prepare an expression vector for hNR10. In the samemanner, an expression vector for cynNR10 was constructed from cynomolgusmonkey NR10 (nucleotide sequence, SEQ ID NO: 65; amino acid sequence,SEQ ID NO: 66).

1.1.2. Preparation of DNA Cartridge

In order to use the hNR10 or cynNR10 expression vector prepared in 1.1.1for DNA immunization of mice, the Helios Gene Gun Cartridge Kit(BIO-RAD) was used to produce a DNA cartridge for each DNA that allowsimmunization with 1 μg of DNA at one time.

1.2. Preparation of Hybridomas Producing Anti-Human NR10 Antibody

1.2.1. Preparation of Hybridomas Using Mice Immunized with Human orCynomolgus Monkey

NR10

Ten Balb/c mice (female; six weeks old at the beginning of immunization;Charles River Laboratories Japan) were immunized with human orcynomolgus monkey NR10 by the following procedure. For primaryimmunization, the mice were immunized with the DNA cartridge preparedwith the hNR10 expression vector using the Helios Gene Gun System(BIO-RAD). One week later, secondary immunization was performed by theHelios Gene Gun System (BIO-RAD) using the DNA cartridge prepared withthe cynNR10 expression vector. The third and subsequent immunizationswere carried out at one-week intervals using the hNR10 and cynNR10expression vectors alternately. After the titer of serum antibodyagainst human NR10 was confirmed to be elevated, a human NR10 protein(extracellular domain) (Referential Example 4) diluted with PBS(−) wasintravenously administered at 10 ng/head as the final immunization. Fourdays after the final immunization, mouse spleen cells were fused withmouse myeloma P3X63Ag8U.1 cells (abbreviated as P3U1; ATCC CRL-1597) bya conventional method using PEG1500 (Roche Diagnostics). The resultingfused cells, i.e., hybridomas, were cultured in RPMI1640 supplementedwith 10% FBS (hereinafter abbreviated as 10% FBS/RPMI1640).

1.2.2. Selection of Hybridomas

On the next day of fusion, the fused cells were suspended in a semisolidmedium (StemCells), and cultured for selection as well as colonizationof hybridomas.

After nine or ten days of fusion, hybridoma colonies were picked up andeach colony was seeded into each well of 96-well plates containing theHAT selection medium (10% FBS/RPMI1640, 2 vol % of HAT 50× concentrate(Dainippon Pharmaceutical), and 5 vol % of BM-Condimed H1 (RocheDiagnostics)). After three to four days of culture, the culturesupernatant was collected from each well to determine the concentrationof mouse IgG in the supernatant. The culture supernatants in which mouseIgG was detected were assessed for a neutralizing activity using a humanIL-31-dependent cell line (hNR10/hOSMR/BaF3 cells; Referential Example2), and several clones having a strong NR10-neutralizing activity wereobtained (FIG. 3). Clones that suppress the human IL-31-induced growthof cells in a concentration-dependent manner and suppress the cynomolgusmonkey IL-31-induced growth of cells (cynNR10/cynOSMR/BaF3 cells;Referential Example 2) in a concentration-dependent manner were obtained(FIG. 4).

[Example 2] Preparation of Chimeric Antibodies Preparation of ExpressionVectors for Chimeric Antibodies

Total RNAs were extracted from the hybridomas using RNeasy Mini Kits(QIAGEN), and cDNAs were synthesized from them using SMART RACE cDNAAmplification Kit (BD Biosciences). Antibody variable region genes wereisolated by PCR using PrimeSTAR HS DNA polymerase (TaKaRa), 10×Universal Primer A Mix attached to SMART RACE cDNA Amplification Kit (BDBiosciences), and primers designed for each antibody constant region (Hchain, mIgG1-rnot; L chain, mIgK-rnot). The nucleotide sequence of eachisolated DNA fragment was determined with ABI PRISM 3730xL DNA Sequenceror ABI PRISM 3700 DNA Sequencer (Applied Biosystems), using BigDyeTerminator Cycle Sequencing Kit (Applied Biosystems) according to themethod described in the appended instruction manual. The determinedamino acid sequences of H chain and L chain variable regions in themouse antibodies NS18, NS22, NS23, and NS33 were shown in FIGS. 1 and 2,respectively.

Each of the resulting H and L chain fragments was subjected to PCR usingPrimeSTAR HS DNA Polymerase (TaKaRa) and the primer sets shown inTable 1. The resulting amplified fragments were ligated with theconstant region (human γ1 or γ2, and human κ, respectively), and theninserted into an animal cell expression vector. The nucleotide sequenceof each DNA fragment was determined with ABI PRISM 3730xL DNA Sequenceror ABI PRISM 3700 DNA Sequencer (Applied Biosystems), using BigDyeTerminator Cycle Sequencing Kit (Applied Biosystems) according to themethod described in the appended instruction manual.

TABLE 1 Sequence (5′ →3′) SEQ ID NO: mIgG1-rnotTAATAGCGGCCGCTCATTATTTACCAGGAGAGTGGGAGAG 90 mIgK-rnotTAATAGCGGCCGCTCATTAACACTCATTCCTGTTGAAGCT 91 mNS18H-fecoGACGAATTCCACCATGGGATGGAGCTGGATCTT 92 mNS18L-fecoGACGAATTCCACCATGAGTGTGCCCACTCAGGT 93 mNS33H-fecoGACGAATTCCACCATGGAATGTAACTGGATACT 94 mNS33L-fecoGACGAATTCCACCATGGATTTTCTGGTGCAGAT 95 Forward primer Reverse primerNS18 H chain mNS18H-feco mIG1-rnot NS18 L chain mNS18L-feco mIGK-rnotNS22 H chain Mns18H-feco mIG1-rnot NS22 L chain mNS18L-feco mIGK-rnotNS23 H chain mNS18H-feco mIG1-rnot NS23 L chain mNS18L-feco mIGK-rnotNS33 H chain mNS33H-feco mIG1-rnot NS33 L chain mNS33L-feco mIGK-rnot

Preparation of Chimeric Antibodies

Human embryonic kidney cancer cell line HEK293H (Invitrogen) wassuspended in DMEM (Invitrogen) supplemented with 10% fetal bovine serum(Invitrogen), and 10 ml of cells were seeded into dishes for adherentcells (10 cm in diameter; CORNING) at a cell density of 6×10⁵ cells/ml.The cells were incubated in a CO₂ incubator (37° C., 5% CO₂) for onewhole day and night. Then, the medium was removed by aspiration, and 6.9ml of CHO-S-SFMII medium (Invitrogen) was added. CHO-S-SFMII medium wasadded to the prepared plasmid DNA mixture (13.8 μg in total) to a volumeof 700 μl. This was mixed with 20.7 μl of 1 μg/ml polyethyleneimine(Polysciences Inc.), and allowed to stand at room temperature for 10minutes. The solution was added to the cells in each dish. The cellswere incubated in a CO₂ incubator (37° C., 5% CO₂) for four to fivehours. Then, 6.9 ml of CHO-S-SFMII medium (Invitrogen) was added, andthe cells were incubated in a CO₂ incubator for three to four days. Theculture supernatants were collected and then centrifuged (approx. 2000g, five minutes, room temperature) to remove the cells. The supernatantswere filtered through 0.22-μm filter MILLEX®-GV (Millipore). Each samplewas stored at 4° C. until use. Antibodies were purified from thesupernatants using Protein G Sepharose (Amersham Biosciences). Thepurified antibodies were concentrated with Amicon Ultra 15 (Millipore),and then the solvent was replaced with PBS(−) containing 0.05% NaN₃using PD-10 Desalting columns (Amersham Biosciences. The absorbance at280 nm was measured with ND-1000 Spectrophotometer (NanoDrop), and theconcentrations were determined by the method of Pace et al. (ProteinScience (1995) 4: 2411-2423).

Assessment of the Activity of Chimeric NS22

The activity of neutralizing hIL-31 was assessed using thehNR10/hOSMR/BaF3 cell line, which grows in an hIL-31 dose-dependentmanner, as described below.

hNR10/hOSMR/BaF3 cells were prepared at 1.5×10⁵ cells/ml using RPMI1640medium (GIBCO) containing 10% FBS (MOREGATE) and 1%Penicillin-Streptomycin (Invitrogen). hIL-31 (R&D Systems) was added toan aliquot of the cells to a final concentration of 4 ng/ml (IL-31(+);final conc.: 2 ng/ml). The remaining cell suspension was used asIL-31(−). The purified NS22 was adjusted to 2 μg/ml using the medium,and eight serial dilutions were prepared at a common dilution ratio of 3(final conc.: 1 μg/ml or less). 50 μl each of the cell suspension andthe dilution of chimeric NS22 (human γ1, κ) was added to each well of96-well flat-bottom plates (CORNING), and the cells were cultured in a5% CO₂ incubator at 37° C. for two days. After culture, 20 μl of amixture of equal amounts of Cell Counting Kit-8 (Dojindo) and PBS wasadded to each well, and the absorbance (450 nm/620 nm) was measured(TECAN, SUNRISE CLASSIC). After the reaction was allowed to continue fortwo hours in a 5% CO₂ incubator at 37° C., the absorbance was measuredagain. The neutralizing activity of NS22 was presented as an inhibitionrate using a value obtained by subtracting the 0-hour value from the2-hour value. The result showed that NS22 suppressed the IL-31-inducedgrowth of the hNR10/hOSMR/BaF3 cell line in a concentration-dependentmanner. This demonstrates that NS22 has a neutralizing activity againstthe human IL-31 signaling (FIG. 5).

The IL-31-neutralizing activity was assessed as described below usingthe DU145 cell line (human prostate cancer cell line), in which IL-6production is induced upon IL-31 stimulation.

DU145 cells were prepared at 2.5×10⁵ cells/ml in MEM (Invitrogen)containing 10% FBS (MOREGATE), 2 mmol/1 L-glutamine (Invitrogen), and 1mmol/1 sodium pyruvate (SIGMA), and 200-μ1 aliquots were dispensed intoeach well of 48-well plates (CORNING). The cells were incubated at 37°C. under 5% CO₂ overnight. The purified chimeric NS22 (human γ1, κ) wasdiluted to 100 μg/ml with MEM containing 10% FBS, 2 mmol/1 L-glutamine,and sodium pyruvate. Using this solution, six serial dilutions wereprepared at a common dilution ratio of 5. Each dilution was combinedwith 100 ng/ml human interleukin-31 (R&D systems) at a ratio of 1:1, anda 50-μl aliquot was added to each well. After two days of culture at 37°C. under 5% CO₂, the concentration of IL-6 in the culture supernatantwas determined using DuoSet ELISA Development kit (R&D systems). Theneutralizing activity of NS22 was assessed by determining the inhibitionrate (%). Specifically, assuming the IL-6 concentration in the absenceof IL-31 (A) as the maximal inhibitory activity (100% inhibition) andthe IL-6 concentration in the presence of IL-31 without NS22 (B) as noinhibitory activity (0% inhibition), the IL-6 concentration in thepresence of IL-31 and NS22 (C) was determined according to the followingformula:

Inhibition rate (%)=(B−C)/(B−A)×100

The result showed that NS22 suppressed the IL-31-induced IL-6 productionin the DU145 cell line in a concentration-dependent manner and thusdemonstrated that NS22 had a neutralizing activity against the humanIL-31 signaling (FIG. 6).

Assessment of Competition of Chimeric Anti-NR10 Antibody with IL-31

Human IL-31 (R&D Systems) was labeled with FMAT Blue MonofunctionalReactive Dye (Applied Biosystems). 100 μl of hIL-31 prepared at 0.5mg/ml using 50 mM sodium phosphate buffer (pH 8.0) was mixed with 5.25μl of 25 nmoles FMAT Blue dissolved in DMSO (Junsei). After vortexing,the mixture was allowed to stand at room temperature for 15 minutes. TheFMAT Blue-conjugating reaction with hIL-31 was terminated by adding 5 μlof 1 M Tris-HCl (pH 7.4) and 1.1 μl of 10% Tween20, and then FMATBlue-labeled hIL-31 and unreacted FMAT Blue were separated by gelfiltration using Superdex 75 (GE Healthcare, 17-0771-01) column with0.1% Tween20/PBS developing solution.

Antibodies were assessed for the activity of inhibiting the IL-31/NR10binding by using hNR10-expressing CHO cells as described below.

NS22 and NA633 (the constant region of each is γ1, κ) were diluted at anappropriate concentration using Assay buffer (10 mM HEPES, 140 mM NaCl,2.5 mM CaCl₂, 3 mM MgCl₂, 2% FBS, 0.01% NaN₃), and then seven serialdilutions were prepared at a common dilution ratio of 2. The dilutionswere added at 40 μl/well to plates (96-Well FMAT Plates; AppliedBiosystems). Then, FMAT Blue-labeled hIL-31 was diluted 400 times withAssay buffer and added at 20 μl/well. Finally, cell suspensions adjustedto 2.5×10⁵ cells/ml using Assay buffer were added at 40 μl/well (final1×10⁴ cells/well). Two hours after addition of cells, the fluorescence(FL1) was determined using the 8200 Cellular Detection System (AppliedBiosystems). The result showed that NS22 inhibited the binding ofhIL-31/hNR10 in a dose-dependent manner, and demonstrated that itsactivity was superior to that of NA633 (FIG. 7).

[Example 3] Competition of Anti-NR10 Antibody Against NR10

The antibody NS22 purified from a hybridoma culture supernatant waslabeled with FMAT Blue (Applied Biosystems, 4328853). 170 μl of NS22prepared at 1 mg/ml in PBS was mixed with 17 μl of 1 M NaHCO₃ solutionand 3.4 μl of FMAT Blue (17 nmoles) dissolved in DMSO. After vortexing,the mixture was allowed to stand at room temperature for 30 minutes. TheFMAT Blue conjugating reaction with NS22 was terminated by adding 8 μlof 1 M Tris-HCl (pH 7.4) and 1.9 μl of 1% Tween 20, and then FMATBlue-labeled NS22 (FMAT Blue-NS22) and unreacted FMAT Blue wereseparated by gel filtration using Superdex 75 (GE Healthcare,17-0771-01) column with 0.01% Tween20/PBS developing solution.

Each antibody was examined for inhibition of the binding of the preparedFMAT Blue-NS22 to hNR10-expressing CHO cells (Referential Example 3)using the 8200 Cellular Detection System (Applied Biosystems, 4342920).The chimeric anti-NR10 antibodies (the constant region of each is γ1, κ)were added at various concentrations to each well containing 7500 cellsand 8.8×10⁻² μg/ml FMAT Blue-NS22. The cells were allowed to stand inthe dark for four hours, and then the fluorescent signal from FMAT Bluebound to the cells was measured. The reaction was carried out in 10 mMHepes-KOH containing 2.5 mM CaCl₂, 3 mM MgCl₂, 140 mM NaCl, 2% FBS, and0.01% NaNO₃. The result is shown in FIG. 8. The fluorescence value FL1,which represents the binding of FMAT Blue-NS22 to NR10-expressing cells,was reduced with the increase in the concentration of antibody NS22 orNS23. On the other hand, FL1 was hardly reduced with the increase in theconcentration of antibody NA633 (Referential Example 6) (FIG. 8).

[Example 4] Humanization of NS22 Antibody

Selection of Each Framework Sequence

The variable regions of mouse NS22 antibody were compared with humangermline sequences. FR sequences used for humanization are summarized inTable 2. CDRs and FRs were determined based on the Kabat numbering. Thehumanized variable region sequences of H chain composed of FR1, FR2,FR3_1, and FR4, and composed of FR1, FR2, FR3_2, and FR4, which arelisted in Table 2, are designated as H0-VH (SEQ ID NO: 50) and H1-VH(SEQ ID NO: 112), respectively. Meanwhile, the sequence of L chaincomposed of FR1, FR2, FR3, and FR4 is designated as L0 (SEQ ID NO: 52).

Preparation of Variable Region for Humanized NS22 H0L0

Synthetic oligo DNAs were designed for each of the H and L chains toconstruct the variable regions of humanized NS22 in which the CDRs ofNS22 are grafted onto the FRs used for humanization. The respectivesynthetic oligo DNAs were mixed, and then subjected to assembly PCR toconstruct a gene encoding the variable region of humanized NS22. Theassembly PCR was carried out using KOD-Plus (TOYOBO) according to thefollowing conditions. A reaction mixture containing 10 pmol syntheticoligo DNAs and the appended PCR Buffer, dNTPs, MgSO₄, and KOD-Plus washeated at 94° C. for five minutes. The mixture was then subjected to twoPCR cycles of 94° C. for two minutes, 55° C. for two minutes, and 68° C.for two minutes. Then, 10 pmol each of a primer in which a restrictionsite and Kozak sequence has been added to the 5′ end of the variableregion, and a primer in which a restriction site has been added to the3′ end of the variable region, was added and subjected to 35 PCR cyclesof 94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for oneminute to yield a amplified fragment. The resulting amplified fragmentwas cloned into TOPO TA Cloning vector (TOYOBO), and its nucleotidesequence was determined by sequencing. The constructed variable regionswere combined with the constant regions to prepare H0-SKSC (SEQ ID NO:54) and L0 (SEQ ID NO: 56). The resulting construct was inserted into anexpression vector capable of expressing the inserted gene in animalcells. The nucleotide sequence of each DNA fragment was determined usingBigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with ABIPRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (AppliedBiosystems) according to the method described in the appendedinstruction manual.

Preparation of Variable Region for Humanized NS22 H1

H1-SKSC (SEQ ID NO: 130) was generated by substituting the glutamine (E)at Kabat-numbering position 73 in FR3 of H0-SKSC (SEQ ID NO: 54) withlysine (K). The mutant was prepared using commercially availableQuikChange Site-Directed Mutagenesis Kit (Stratagene) according to theappended instruction manual.

Expression of IgG-Converted Antibody

Antibody expression was performed by the method described below. Humanfetal renal cancer cell-derived cell line HEK293H (Invitrogen) wassuspended in DMEM (Invitrogen) containing 10% fetal bovine serum(Invitrogen), and 10 ml of cells at a density of 5-6×10⁵ cells/ml wasseeded onto dishes for adherent cells (10 cm in diameter; CORNING). Thecells were incubated in a CO₂ incubator (37° C., 5% CO₂) for one wholeday and night. Then, the medium was removed by aspiration, and 6.9 ml ofCHO-S-SFMII medium (Invitrogen) was added to the cells. The preparedplasmid DNA mixture (13.8 μg in total) was mixed with 20.7 μl of 1 μg/mlpolyethyleneimine (Polysciences Inc.) and 690 μl of CHO-S-SFMII medium,and allowed to stand at room temperature for 10 minutes. The mixture wasadded to the cells in each dish, and the cells were incubated in a CO₂incubator (5% CO₂, 37° C.) for four to five hours. Then, 6.9 ml ofCHO-S-SFMII medium (Invitrogen) was added, and the cells were incubatedin a CO₂ incubator for three days. The culture supernatant was collectedand centrifuged (approx. 2000 g, five minutes, room temperature) toremove the cells. The supernatant was then sterilized by filtrationthrough 0.22-μm filter MILLEX®-GV (Millipore). Each sample was stored at4° C. until use.

Purification of IgG-Converted Antibody

50 μl of rProtein A Sepharose™ Fast Flow (Amersham Biosciences)suspended in TBS was added to the obtained culture supernatant, andmixed by inversion at 4° C. for four hours or more. The solution wastransferred to 0.22-μm filter cup of Ultrafree®-MC (Millipore). Afterthree washes with 500 μl of TBS, rProtein A Sepharose™ resin wassuspended in 100 μl of aqueous solution of 50 mM sodium acetate (pH3.3), and allowed to stand for three minutes to elute the antibody. Thesolution was immediately neutralized by adding 6.7 μl of 1.5 M Tris-HCl(pH 7.8). The elution was performed twice and 200 μl of purifiedantibody was obtained. 2 μl of the antibody-containing solution wassubjected to ND-1000 Spectrophotometer (NanoDrop)(Thermo ScientificNanoDrop™ 1000 Spectrophotometer (Thermo Scientific)) or 50 μl wassubjected to Spectrophotometer DU-600 (BECKMAN) to measure absorbance at280 nm, and the antibody concentration was calculated by the method ofPace et al. (Protein Science (1995) 4: 2411-2423).

Measurement of Competition with IL-31 Using FMAT

Antibodies were assessed for the activity of inhibiting the IL-31/NR10binding by using hNR10-expressing CHO cells as described below. Thechimeric NS22 antibody and NS22_H0L0 (H chain, H0-SKSC/SEQ ID NO: 54; Lchain, L0/SEQ ID NO: 56) were diluted at an appropriate concentrationusing Assay buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, 3 mM MgCl₂,2% FBS, 0.01% NaN₃, pH7.4), and further eight serial dilutions wereprepared at a common dilution ration of 2. The dilutions were added at40 μl/well to plates (96-Well FMAT Plates, Applied Biosystems). Then,FMAT Blue-labeled hIL-31 was diluted 400 times with Assay buffer, andadded at 20 μl/well. Finally, a cell suspension adjusted to 2.5×10⁵cells/ml using Assay buffer was added at 40 μl/well (final 1×10⁴cells/well). Two hours after addition of cells, the fluorescence (FL1)was measured using the 8200 Cellular Detection System (AppliedBiosystems).

The result showed that, as shown in FIG. 9, humanized NS22 antibodiesH0L0 (H chain, H0-SKSC/SEQ ID NO: 54; L chain, L0/SEQ ID NO: 56), andH1L0 (H chain, H1-SKSC/SEQ ID NO: 130; L chain, L0/SEQ ID NO: 56)exhibited a competition activity comparable to that of the chimericantibody, suggesting that both H0L0 and H1L0 are humanized anti-IL-31receptor antibodies. In addition, it is considered that the FRs used forH0L0 and H1L0 can be used for humanization.

Accordingly, all of the mutations in CDRs described in the Exampleshereinafter can be introduced into both H0 and H1.

TABLE 2 HO Germline Human FR sequence FR1Germline: hVH_1_46(Accession No. X92343) QVQLVQSGAEVKKPGASVKVSCKASGYTFT(SEQ ID NO: 96) FR2 Germline: hVH_1_46 (Accession No. X92343)WVRQAPGQGLEWMG (SEQ ID NO: 97) FR3_1Germline: hVH_1_69 (Accession No. L22582)RVTITADESTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 98) FR3_2Germline: hVH_1_69 (Accession No. Z27506)RVTITADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 131) FR4 Germline: JH1WGQGTLVTVSS (SEQ ID NO: 99) LO Germline Human FR sequence FR1Germline: hVK_1_39 (Accession No. X59315) DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO: 100) FR2 Germline : hVK_1_39 (Accession No. X59315)WYQQKPGKAPKLLIY (SEQ ID NO: 101) FR3Germline: hVK_1_39 (Accession No. X59315)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 102) FR4 Germline: JK4FGGGTKVEIK (SEQ ID NO: 103)

[Example 5] Heterogeneity-Reducing Effect of Novel Constant Regions M14and M58 in Humanized Anti-IL31 Receptor Antibody

As shown in Referential Examples 7 to 9, it was demonstrated that theconversion of the constant region from IgG2 to M14 or M58 in the huPM1antibody, a humanized anti-IL-6 receptor antibody, could reduce theheterogeneity derived from the IgG2 hinge region without loss ofstability. Thus, humanized anti-IL-31 receptor antibodies were alsotested to assess whether the heterogeneity can be reduced by convertingtheir constant regions from the wild-type IgG2 to M14 or M58.

H0-M14, H0-M58, H0-IgG1, and H0-IgG2, which were generated by combiningIgG1 (SEQ ID NO: 60), IgG2 (SEQ ID NO: 132), M14 (SEQ ID NO: 129) andM58 (SEQ ID NO: 128) generated in Referential Examples 8 and 9, with Hchain variable region H0 (H0-VH/SEQ ID NO: 50) of humanized anti-IL-31receptor antibody generated in Example 4, were used as H chains, and L0(L0/SEQ ID NO: 56) produced in Example 4 was used as an L chain, togenerate H0L0-IgG1 (H chain, H0-IgG1/SEQ ID NO: 133; L chain, L0/SEQ IDNO: 56), H0L0-IgG2 (H chain, H0-IgG2/SEQ ID NO: 134; L chain, L0/SEQ IDNO: 56), H0L0-M14 (H chain, H0-M14/SEQ ID NO: 135; L chain, L0/SEQ IDNO: 56), and H0L0-M58 (H chain, H0-M58/SEQ ID NO: 136; L chain, L0/SEQID NO: 56). Each antibody was expressed and purified by the methoddescribed in Example 4.

The heterogeneity was assessed by cation exchange chromatography. Theprepared antibodies were assessed for heterogeneity using ProPac WCX-10(Dionex) column, 20 mM sodium acetate (pH 5.0) as mobile phase A, and 20mM sodium acetate/1M NaCl (pH 5.0) as mobile phase B, with anappropriate flow rate and gradient. The result of assessment by cationexchange chromatography (IEC) is shown in FIG. 10.

As shown in FIG. 10, the heterogeneity was increased by conversion ofthe constant region from IgG1 to IgG2 in the anti-IL-31 receptorantibody, and the heterogeneity can be reduced by conversion of theconstant region to M14 or M58 in any antibody.

[Example 6] Pharmacokinetics Improving Effect of Novel Constant RegionM58 in Anti-IL-31 Receptor Antibodies

As shown in Referential Example 9, conversion of the constant regionfrom IgG1 to M58 in anti-IL-6 receptor antibody huPM1 was found toimprove its human FcRn-binding activity and the pharmacokinetics inhuman FcRn transgenic mice. Thus, anti-IL-31 receptor antibodies werealso tested to assess whether conversion of the constant region to M58improves their pharmacokinetics.

H0L0-IgG1 (H chain: H0-IgG1/SEQ ID NO: 133; L chain: L0/SEQ ID NO: 56)and H0L0-M58 (H chain: H0-M58/SEQ ID NO: 136; L chain L0/SEQ ID NO: 56)prepared as described in Examples 4 and 5 were assessed for the humanFcRn-binding activity by the method described in Referential Example 9.The result is shown in Table 3.

TABLE 3 KD (μM) H0L0-IgG1 1.07 H0L0-M58 0.91

As shown in Table 3, conversion of the constant region from IgG1 to M58also improved the human FcRn-binding activity of the anti-IL-31 receptorantibody H0L0 as in the anti-IL-6 receptor antibody hPM1. This suggeststhat conversion of the constant region from IgG1 to M58 may improve thepharmacokinetics of anti-IL-31 receptor antibody in human.

[Example 7] Identification of Mutation Sites Reducing the IsoelectricPoint Production of Mutants

Each mutant was produced by the method described in Example 4 or byassembly PCR. In the method using assembly PCR, oligo DNAs aresynthesized based on forward and reverse sequences including an alteredsite. Forward oligo DNA including an altered site and reverse oligo DNAbinding to the vector in which the gene to be altered was inserted werecombined, and reverse oligo DNA including an altered site and forwardoligo DNA binding to the vector in which the gene to be altered wasinserted were combined. PCR was carried out using PrimeSTAR (Takara) toproduce 5′-end and 3′-end fragments including the altered site. The twofragments were assembled by assembly PCR to produce each mutant. Theproduced mutant was inserted into an expression vector capable ofexpressing the insert gene in animal cells. The nucleotide sequence ofthe resulting expression vector was determined by a method known tothose skilled in the art. Antibodies were produced and purified by themethod described in Example 4.

Identification of Mutation Sites

To improve the pharmacokinetics of H0L0 (H chain, H0-SKSC/SEQ ID NO: 54;L chain, L0/SEQ ID NO: 56), altered sites capable of reducing theisoelectric point of the variable region were examined. Screening ofmutation sites in the variable regions predicted from thethree-dimensional structure model revealed mutation sites that woulddecrease the isoelectric point of the variable regions withoutsignificantly reducing its binding to NR10. These are summarized inTable 4 (Hp5-VH/SEQ ID NO: 137, Hp7-VH/SEQ ID NO: 138, Hp8-VH/SEQ ID NO:139, Hp6-VH/SEQ ID NO: 140, Hp9-VH/SEQ ID NO: 141, Hp1-VH/SEQ ID NO:142, Hp13-VH/SEQ ID NO: 143, Lp1-VL/SEQ ID NO: 144, Lp2-VL/SEQ ID NO:145, Lp3-VL/SEQ ID NO: 146, Lp4-VL/SEQ ID NO: 147, Lp7-VL/SEQ ID NO:148, Lp5-VL/SEQ ID NO: 149, Lp6-VL/SEQ ID NO: 150). Each variant wasproduced and purified by the method described in Example 4.

Each variant was tested for the activity of inhibiting the hIL-31/hNR10binding by using FMAT. The test was carried out according to the methodas described in Example 4. As shown in FIG. 11, the competition activityof each variant was not greatly reduced as compared to that of H0L0.

TABLE 4 Mutation site H0 Amino acid Sequence after Name Type H0 sequence(kabat No) sequence after mutation mutation Hp5 FR2 WVRQAPGQGLEWMG 38 RQ WVQQSPGQGLEWMG (SEQ ID NO: 97) 40 *A S (SEQ ID NO: 120) Hp7 CDR2LINPYNGGTSYNQKFKG 50 L E EINPYNGGTSYNQKFKG (SEQ ID NO: 10)(SEQ ID NO: 113) Hp8 CDR2 LINPYNGGTSYNQKFKG 52 N D LIDPYNGGTSYNQKFKG(SEQ ID NO: 10) (SEQ ID NO: 114) Hp6 CDR2 LINPYNGGTSYNQKFKG 61 Q DLINPYNGGTSYNDKFKG (SEQ ID NO: 10) (SEQ ID NO: 115) Hp9 CDR2LINPYNGGTSYNQKFKG 62 K Q LINPYNGGTSYNQQFKG (SEQ ID NO: 10)(SEQ ID NO: 116) Hp1 CDR2 LINPYNGGTSYNQKFKG 64 K Q LINPYNGGTSYNQKFQG(SEQ ID NO: 10) (SEQ ID NO: 117) Hp13 CDR2 LINPYNGGTSYNQKFKG 64 K QLINPYNGGTSYNQKFQD (SEQ ID NO: 10) 65 G D (SEQ ID NO: 119) Mutation siteL0 Amino acid Sequence after Name Type L0 sequence (kabat No) sequenceafter mutation mutation Lp1 CDR1 RTSENIYSFLA 24 R Q QTSENIYSFLA(SEQ ID NO: 13) (SEQ ID NO: 121) Lp2 CDR1 RTSENIYSFLA 28 N D RTSEDIYSFLA(SEQ ID NO: 13) (SEQ ID NO: 122) Lp3 CDR2 NAKTLAK 50 N D DAKTLAK(SEQ ID NO: 14) (SEQ ID NO: 123) Lp4 CDR2 NAKTLAK 52 K Q NAQTLAK(SEQ ID NO: 14) (SEQ ID NO: 124) Lp7 CDR2 NAKTLAK 54 L E NAKTEAK(SEQ ID NO: 14) (SEQ ID NO: 125) Lp5 CDR2 NAKTLAK 56 K Q NAKTLAQ(SEQ ID NO: 14) (SEQ ID NO: 126) Lp6 CDR2 NAKTLAK 56 K D NAKTLAD(SEQ ID NO: 14) (SEQ ID NO: 127)

Asterisk (*) in Table 4 above indicates a site that was not relevant tothe isoelectric point but altered for conversion into a human sequence.

Examples of the humanized NS22 antibodies whose isoelectric point hasbeen reduced by combining these alterations include Hp3Lp15 (H chain:Hp3-SKSC/SEQ ID NO: 151; L chain: Lp15/SEQ ID NO: 152). Affinity forNR10, isoelectric point, and plasma retention in mice were comparedbetween Hp3Lp15 and H0L0.

Measurement of Affinity

The affinity of each antibody for NR10 was determined by the methoddescribed in Referential Example 10.

The result of affinity measurement is shown in Table 5. The affinity ofHp3Lp15 was shown to be almost the same as that of H0L0.

TABLE 5 ka (1/Ms) kd (1/s) KD (M) H0L0 3.7E+05 1.2E−03 3.3E−09 Hp3Lp154.2E+05 1.6E−03 3.9E−09

Measurement of Isoelectric Point

Each antibody was analyzed by isoelectric focusing to assess changes inthe isoelectric point of the whole antibody due to the amino acidalterations in its variable region. Isoelectric focusing was performedby the following method.

Phast-Gel Dry IEF gel (Amersham Biosciences) was swollen in PhastsystemCassette (Amersham Biosciences) for about 30 minutes using the swellingsolution shown below.

MilliQ water 1.5 ml

Pharmalyte 5-8 for IEF (Amersham Biosciences) 100 μl

Electrophoresis was carried out in PhastSystem (Amersham Biosciences)using the swollen gel according to the program indicated below. Thesamples were loaded onto the gel in Step 2. Calibration Kit for pI(Amersham Biosciences) was used as a pI marker.

Step 1: 2000 V 2.5 mA 3.5 W 15° C. 75 Vh

Step 2: 200 V 2.5 mA 3.5 W 15° C. 15 Vh

Step 3: 2000 V 2.5 mA 3.5 W 15° C. 410 Vh

After electrophoresis, the gel was fixed with 20% TCA, and thensilver-stained using the Silver Staining Kit, Protein (AmershamBiosciences), according to the protocol attached to the kit. Afterstaining, the isoelectric point of the sample (the whole antibody) wascalculated from the known isoelectric points of the pI markers.

The result of isoelectric point measurement by isoelectric focusingshowed that the isoelectric point of H0L0 was about 7.8, and theisoelectric point of Hp3Lp15 was about 5.5, showing that the isoelectricpoint of Hp3Lp15 was decreased by about 2.3 as compared to H0L0. Whenthe theoretical isoelectric point of the variable region VH/VL wascalculated by GENETYX (GENETYX CORPORATION), the theoretical isoelectricpoints of the variable regions of H0L0 and Hp3Lp15 were 7.76 and 4.63,respectively. Thus, the theoretical isoelectric point of Hp3Lp15 wasdecreased by 3.13 as compared to H0L0.

Assessment of Pharmacokinetics of Antibody with Reduced IsoelectricPoint Using Mice

In order to assess the plasma retention of Hp3Lp15, a modified antibodywith reduced isoelectric point, the plasma retention of H0L0 and Hp3Lp15was compared in normal mice. A single dose of H0L0 or Hp3Lp15 wasintravenously administered at 1 mg/kg to mice (C57BL/6J, Charles RiverJapan, Inc.) to compare the time course of the plasma concentration. Theplasma concentrations were determined by ELISA. Appropriateconcentrations of a calibration sample and test plasma samples weredispensed into immunoplates (Nunc-Immuno Plate, MaxiSorp (Nalge NuncInternational)) coated with anti-human IgG (Fc-specific) antibody(Sigma). The samples were allowed to stand at room temperature for onehour. After reaction with Goat Anti-Human IgG-ALP (Sigma) at roomtemperature for one hour, color developing reaction was carried outusing BluePhos Microwell Phosphatase Substrates System (Kirkegaard &Perry Laboratories) as a substrate. The absorbance at 650 nm wasmeasured with a microplate reader. The plasma concentrations weredetermined based on the absorbance of the calibration curve using theanalytical software SOFTmax PRO (Molecular Devices).

Pharmacokinetic parameters (AUC and systemic clearance (CL)) werecalculated from the obtained time-course data of the plasmaconcentration using the pharmacokinetics analysis software WinNonlin(Pharsight). The parameters are shown in Table 6. AUC and the clearanceof Hp3Lp15 after the intravenous administration were reduced by about14% and about 12%, respectively, as compared to H0L0. Thus, it wasdemonstrated that Hp3Lp15, in which the isoelectric point of H0L0 hasbeen reduced, had improved pharmacokinetics.

TABLE 6 AUC CL (μg · d/kg) (ml/d/kg) Mean SD Mean SD H0L0 281.8 13.1 3.60.2 Hp3Lp15 321.1 26.1 3.1 0.3

[Example 8] Effect of Combinations of Variable Region and ConstantRegion on the Biological Activity

In order to assess the effects of different constant regions on thebiological activity, the following variants were produced.

SKSC (SEQ ID NO: 62) and M58 (SEQ ID NO: 128), constant regions preparedin Referential Examples 7 and 9, were combined with Hp3 (Hp3-VH/SEQ IDNO: 167), a variable region prepared in Example 7, to produce Hp3-M58(SEQ ID NO: 240) and Hp3-SKSC (SEQ ID NO: 151) as H chains. The preparedH chains were combined with Lp15 (Lp15/SEQ ID NO: 152), an L chainprepared in Example 7, to produce Hp3Lp15-SKSC (H chain, Hp3-SKSC/SEQ IDNO: 151; L chain, Lp15/SEQ ID NO: 152) and Hp3Lp15-M58 (H chain,Hp3-M58/SEQ ID NO: 240; L chain, Lp15/SEQ ID NO: 152). Each antibody wasexpressed and purified by the method described in Example 4.

The antibodies produced as described above, H0L0-SKSC (H chain,H0-SKSC/SEQ ID NO: 54; L chain, L0/SEQ ID NO: 56) prepared using theconstant region SKSC (SEQ ID NO: 62) described in Referential Example 7,and H0L0-M58 (H chain, H0-M58/SEQ ID NO: 136; L chain, L0/SEQ ID NO: 56)and H0L0-IgG2 (H chain, H0-IgG2/SEQ ID NO: 134; L chain, L0/SEQ ID NO:56) prepared in Example 5, were assessed for the biological activity bythe method described in Example 2 using BaF/NR10. The result issummarized in FIG. 18.

As shown in FIG. 18, no significant difference in the biologicalactivity was detected between the constant regions. Since the biologicalactivity was not affected when combining the two variable regions H0 andHp3 with each constant region, combining variable regions created infuture with any constant region would not result in alteration in thebiological activity.

[Example 9] Identification of Mutation Sites Suppressing Degradation byThermal Acceleration Study

Antibodies used for pharmaceuticals have heterogeneity even though theyare monoclonal antibodies obtained from clones derived from singleantibody-producing cells. Such antibody heterogeneity is known to resultfrom modification such as oxidation or deamidation, and to be increasedduring long-term storage or upon exposure to stress conditions, such asheat stress or light stress (see “Heterogeneity of MonoclonalAntibodies”, Journal of pharmaceutical sciences, vol. 97, No. 7,2426-2447). However, when an antibody is developed as a pharmaceutical,physical properties of the protein, particularly homogeneity andstability, are highly important. Thus, it is desired that theheterogeneity of desired/related substances be reduced and the substancebe composed of a single substance as much as possible. In this context,the experiment described below was conducted to assess the antibodyheterogeneity under stress conditions and to reduce the heterogeneity.

To assess degradation products, an accelerated sample of H0L0 (H chain,H0-SKSC/SEQ ID NO: 54; L chain, L0/SEQ ID NO: 56) was prepared by themethod described below. The prepared accelerated sample andnon-accelerated sample (initial) were analyzed by cation exchangechromatography using the method described below.

Method for preparing accelerated samples

Buffer: PBS

Antibody concentration: 0.2 to 1.0 mg/ml

Acceleration temperature: 60° C.

Acceleration period: one day

Method for analysis by cation exchange chromatography

Column: ProPac WCX-10, 4×250 mm (Dionex)

Mobile phase: (A) 25 mmol/1 MES/NaOH, pH 6.1

-   -   (B) 25 mmol/1 MES/NaOH, 250 mmol/l NaCl, pH 6.1

Flow rate: 0.5 ml/min

Column temperature: 40° C.

Gradient: % B 0 to 0 (0-5 min)→0 to 30 (5-80 min)

Detection: 280 nm

The resulting chromatograms for H0L0 samples before and afteracceleration are shown in FIG. 19. The H0L0 sample after accelerationhad a tendency to show an increased basic peak.

Then, screening was carried out to reduce this peak. As a result, Ha355,Ha356, Ha360, and Ha362 were found. These H chain variants were combinedwith L0 to produce Ha355L0 (H chain, Ha355-SKSC/SEQ ID NO: 242; L chain,L0/SEQ ID NO: 56), Ha356L0 (H chain, Ha356-SKSC/SEQ ID NO: 243; L chain,L0/SEQ ID NO: 56), Ha360L0 (H chain, Ha360-SKSC/SEQ ID NO: 244; L chain,L0/SEQ ID NO: 56), and Ha362L0 (H chain, Ha362-SKSC/SEQ ID NO: 245; Lchain, L0/SEQ ID NO: 56). The sequence of each variant is shown in Table7.

TABLE 7 Mutation site H0 Amino acid Sequence after Name Type H0 sequence(kabat No) sequence after mutation mutation Ha355 CDR3 DGYDDGPYTMDY 100dM L DGYDDGPYTLET (SEQ ID NO: 265) 101 D E (SEQ ID NO: 266) 102 Y T Ha356CDR3 DGYDDGPYTMDY 101 D E DGYDDGPYTMET (SEQ ID NO: 265) 102 Y T(SEQ ID NO: 267) Ha360 CDR3 DGYDDGPYTMDY 97 Y L DGLDDGPYTMET(SEQ ID NO: 265) 101 D E (SEQ ID NO: 268) 102 Y T Ha362 CDR3DGYDDGPYTMDY 97 Y L DGLDDGPYTMES (SEQ ID NO: 265) 101 D E(SEQ ID NO: 269) 102 Y S

Each of the identified antibodies was expressed and purified by themethod described in Example 4. As with H0L0, a accelerated sample ofeach prepared antibody was prepared, and analyzed by cation exchangechromatography. The result is shown in FIG. 19.

The result showed that the generation of the basic peak increased afteracceleration was reduced in the modified antibody containing asubstitution of aspartic acid with glutamic acid at position 101 in theH chain, as compared to H0L0. The modified antibodies were assessed forthe biological activity by the method described in Example 2 usingBaF/NR10. The result is shown in FIG. 20. As shown in FIG. 20, thebiological activities of the modified antibodies were comparable to orstronger than that of H0L0. These findings demonstrated that themodifications of Ha355, Ha356, Ha360, and Ha362 suppressed thegeneration of degradation products by acceleration, and therefore areeffective in improving the stability of antibody.

[Example 10] Identification of Mutation Sites Increasing the Affinity

A library in which mutations were introduced into CDR sequences wasconstructed and examined to improve the affinity of H0L0 for NR10. As aresult of screening of the library in which mutations were introducedinto CDRs, mutations that improve the affinity for NR10 were found. Themutations are shown in Table 8. Each of the H chain variants Ha101-SKSC(SEQ ID NO: 246), Ha103-SKSC (SEQ ID NO: 247), Ha111-SKSC (SEQ ID NO:248), Ha204-SKSC (SEQ ID NO: 249), and Ha219-SKSC (SEQ ID NO: 250) wascombined with L0 (L0/SEQ ID NO: 56); and each of the modified L chainsLa134 (SEQ ID NO: 251), La130 (SEQ ID NO: 252), La303 (SEQ ID NO: 253),and La328 (SEQ ID NO: 254) was combined with H0 (H0-SKSC/SEQ ID NO: 54),to construct an antibody. Each variant was produced and purified by themethod described in Example 4.

The affinity of each antibody for NR10 was assessed using Biacore. Theresult is shown in Table 9. The assay was carried out using the methoddescribed in Referential Example 10. As shown in Table 9, the KD valuefor each variant was found to be improved as compared to that of H0L0 (Hchain, H0-SKSC/SEQ ID NO: 54; L chain, L0/SEQ ID NO: 56).

TABLE 8 Mutation site H0 Amino acid Sequence Name Type H0 sequence(kabat No) sequence after mutation after mutation Ha101 CDR1 GYIMN 33 IV GYVMN (SEQ ID NO: 270) (SEQ ID NO: 272) Ha103 CDR1 GYIMN 34 M I GYIIN(SEQ ID NO: 270) (SEQ ID NO: 273) Ha111 CDR1 GYIMN 34 M L GYILN(SEQ ID NO: 270) (SEQ ID NO: 274) Ha204 CDR2 LINPYNGGTSYNQKFKG 58 S DLINPYNGGTDYNQKFKG (SEQ ID NO: 271) (SEQ ID NO: 275) Ha219 CDR2LINPYNGGTSYNQKFKG 61 Q P LINPYNGGTSYNPKFKG (SEQ ID NO: 271)(SEQ ID NO: 276) Mutation site L0 Amino acid Sequence Name TypeL0 sequence (kabat No) sequence after mutation after mutation La134 CDR1RTSENIYSFLA 31 S R RTSENIYRFLA (SEQ ID NO: 277) (SEQ ID NO: 279) La130CDR1 RTSENIYSFLA 31 S R RTSENIYRFVA (SEQ ID NO: 277) 33 L V(SEQ ID NO: 280) Ls303 CDR3 QHHYESPLT 93 E D QHHYDSPLT (SEQ ID NO: 278)(SEQ ID NO: 281) La328 CDR3 QHHYESPLT 94 S D QHHYEDPLT (SEQ ID NO: 278)(SEQ ID NO: 282) La326 CDR3 QHHYESPLT 97 T F QHHYESPLF (SEQ ID NO: 278)(SEQ ID NO: 283)

TABLE 9 Name ka (1/Ms) kd (1/s) KD (M) H0L0 1.9E+05 6.2E−04 3.2E−09Ha101L0 2.0E+05 3.1E−04 1.5E−09 Ha103L0 2.2E+05 5.3E−04 2.4E−09 Ha111L02.6E+05 5.6E−04 2.1E−09 Ha204L0 3.7E+05 4.8E−04 1.3E−09 Ha219L0 3.2E+059.6E−04 3.0E−09 H0L0 1.5E+05 7.4E−04 5.1E−09 H0La134 2.5E+05 4.4E−041.8E−09 H0La130 2.6E+05 4.0E−04 1.5E−09 H0La303 2.2E+05 4.6E−04 2.1E−09H0La328 1.8E+05 5.2E−04 2.9E−09 H0La326 1.4E+05 5.2E−04 3.7E−09

Examples of combinations of these affinity-improving mutations with theisoelectric point-lowering mutations generated in Example 7 include, forexample, Ha401La402 (H chain, Ha401-SKSC/SEQ ID NO: 255; L chain,La402/SEQ ID NO: 256) and H17L11 (H chain, H17-M58/SEQ ID NO: 222; Lchain, L11/SEQ ID NO: 236). Each variant was produced and purified bythe method described in Example 4.

Ha401La402 (H chain, Ha401-SKSC/SEQ ID NO: 255; L chain, La402/SEQ IDNO: 256) was assessed for its affinity for NR10 and its biologicalactivity by the method described in Referential Example 10 and themethod using BaF/NR10 as described in Example 2, respectively, and theywere compared to those of H0L0 (H chain, H0-SKSC/SEQ ID NO: 54; L chain,L0/SEQ ID NO: 56). The result of affinity measurement is shown in Table10, and the biological activity determined using BaF/NR10 is shown inFIG. 21. Both affinity and biological activity were found to be improvedas compared to those of H0L0 (H chain, H0-SKSC/SEQ ID NO: 54; L chain,L0/SEQ ID NO: 56).

TABLE 10 ka (1/Ms) kd (1/s) KD (M) H0L0 2.9E+05 9.1E−04 3.2E−09Ha401La402 5.8E+05 2.9E−04 5.0E−10

Furthermore, H17L11 (H chain, H17-M58/SEQ ID NO: 222; L chain, L11/SEQID NO: 236) was assessed for its affinity for NR10 and its biologicalactivity by the method described in Example 7 and the method usingBaF/NR10 as described in Example 2, respectively, and they were comparedto those of H0L0 (H chain, H0-M58/SEQ ID NO: 136; L chain, L0/SEQ ID NO:56). The result of affinity measurement is shown in Table 11, and thebiological activity determined using BaF/NR10 is shown in FIG. 22. Bothaffinity and biological activity were found to be improved as comparedto those of H0L0 (H chain, H0-M58/SEQ ID NO: 136; L chain, L0/SEQ ID NO:56).

TABLE 11 ka (1/Ms) kd (1/s) KD (M) H0L0 1.4E+05 6.9E−04 4.8E−09 H17L114.3E+05 2.6E−04 6.2E−10

[Example 11] Identification of Mutation Sites Reducing ImmunogenicityRisk Reduction of Immunogenicity Risk in H Chain CDR1

T-cell epitopes present in the variable region sequence of H0L0 wereanalyzed using TEPITOPE (Methods 2004 December; 34(4): 468-75). As aresult, CDR1 of the H chain was predicted to have many T-cell epitopesthat bind to HLA (i.e. have sequences with a high immunogenicity risk).Then, TEPITOPE analysis was carried out to examine substitutions thatwould reduce the immunogenicity risk of the H chain CDR1. As a result,the immunogenicity risk was found to be greatly reduced by substitutingisoleucine at position 33 in kabat numbering with alanine (A) (Table12). Then, this alteration was added to H17 generated in Example 10 toproduce H19 (H19-M58/SEQ ID NO: 223). The generated H19 was combinedwith L12 to produce H19L12 (H chain, H19-M58/SEQ ID NO: 223; L chain,L12/SEQ ID NO: 237). Each variant was produced and purified by themethod described in Example 4.

The antibody was assessed for the affinity for NR10 and the biologicalactivity by the method described in Referential Example 10 and themethod using BaF/NR10 as described in Example 2, respectively, and theywere compared to those of H0L0 (H chain, H0-M58/SEQ ID NO: 136; L chain,L0/SEQ ID NO: 56). The result of affinity measurement is shown in Table13, and the biological activity determined using BaF/NR10 is shown inFIG. 23. Both affinity and biological activity were shown to be almostequal to those of H0L0.

TABLE 12 Mutation site H0 Amino acid Sequence Name Type H0 sequence(kabat No) sequence after mutation after mutation H19 CDR1 GYIMN 33 I AGYAMN (SEQ ID NO: 270) (SEQ ID NO: 284)

TABLE 13 ka (1/Ms) kd (1/s) KD (M) H0L0 1.8E+05 8.7E−04 4.8E−09 H19L122.3E+05 1.2E−03 5.1E−09

Reduction of Immunogenicity Risk in L Chain CDR1

Threonine (T) present at kabat-numbering position 25 in CDR1 of the Lchain corresponds to alanine (A) or serine (S) in the germline sequence.Thus, it is predicted that the immunogenicity risk is reduced bysubstituting threonine (T) at position 25 with alanine (A) or serine (S)(Table 14). Therefore, the above substitution was added to L12 toproduce L17 (SEQ ID NO: 238). The produced L17 was combined with H0 toproduce H0L17 (H chain, H0-M58/SEQ ID NO: 136; L chain, L17/SEQ ID NO:238). Each variant was produced and purified by the method described inExample 4.

Each variant was assessed for the affinity for NR10 and the biologicalactivity by the method described in Referential Example 10 and themethod using BaF/NR10 as described in Example 2, respectively, and theywere compared to those of H0L0 (H chain, H0-M58/SEQ ID NO: 136; L chain,L0/SEQ ID NO: 56) and H0L12 (H chain, H0-M58/SEQ ID NO: 136; L chain,L12/SEQ ID NO: 237). Since L12 contains a sequence that improves theaffinity, it exhibits about two times higher affinity than H0L0. Theresult of affinity measurement is shown in Table 15, and the biologicalactivity determined using BaF/NR10 is shown in FIG. 24. Both affinityand biological activity were shown to be almost equal to those of H0L12.

TABLE 14 Mutation site L0 Amino acid Sequence Name Type L0 sequence(kabat No) sequence after mutation after mutation Ld-1 CDR1 RTSENIYSFLA25 T A RASENIYSFLA (SEQ ID NO: 277) (SEQ ID NO: 285) Ld-2 CDR1RTSENIYSFLA 25 T S RSSENIYSFLA (SEQ ID NO: 277) (SEQ ID NO: 286)

TABLE 15 ka (1/Ms) kd (1/s) KD (M) H0L0 1.6E+05 7.8E−04 4.8E−09 H0L123.8E+05 7.4E−04 2.0E−09 H0L17 3.9E+05 8.1E−04 2.1E−09

[Example 12] Preparation of Completely Humanized NS22 Antibody

Variable regions of NS22 variants were prepared by combining themultiple mutations that reduce the pI, increase the affinity, suppressthe degradation of H chain, and reduce the immunogenicity risk, all ofwhich were found in the above Examples, in H0 (H0-M58/SEQ ID NO: 136),H1 (H1-M58/SEQ ID NO: 257), or L0 (L0/SEQ ID NO: 56), and subjected tovarious screening procedures. As a result, H28L17 (H chain, H28-M58/SEQID NO: 224; L chain, L17/SEQ ID NO: 238), H30L17 (H chain, H30-M58/SEQID NO: 225; L chain, L17/SEQ ID NO: 238), H34L17 (H chain, H34-M58/SEQID NO: 226, L chain, L17/SEQ ID NO: 238), H42L17 (H chain, H42-M58/SEQID NO: 227; L chain, L17/SEQ ID NO: 238), H44L17 (H chain, H44-M58/SEQID NO: 228; L chain, L17/SEQ ID NO: 238), H46L17 (H chain, H46-M58/SEQID NO: 229; L chain, L17/SEQ ID NO: 238), H57L17 (H chain, H57-M58/SEQID NO: 230; L chain, L17/SEQ ID NO: 238), H71L17 (H chain, H71-M58/SEQID NO: 231; L chain, L17/SEQ ID NO: 238), H78L17 (H chain, H78-M58/SEQID NO: 232; L chain, L17/SEQ ID NO: 238), H92L17 (H chain, H92-M58/SEQID NO: 233; L chain, L17/SEQ ID NO: 238), H97L50 (H chain, H97-M58/SEQID NO: 234; L chain, L50/SEQ ID NO: 239), and H98L50 (H chain,H98-M58/SEQ ID NO: 235; L chain, L50/SEQ ID NO: 239) were found. Eachvariant was produced and purified by the method described in Example 4.

Each variant was assessed for the affinity for NR10 and the biologicalactivity by the method described in Referential Example 10 and themethod using BaF/NR10 as described in Example 2, respectively, and theywere compared to those of H0L0 (H chain, H0-M58/SEQ ID NO: 136; L chain,L0/SEQ ID NO: 56). The result of affinity measurement is shown in Table16, and the biological activity determined using BaF/NR10 is shown inFIGS. 25-1 and 25-2. Both affinity and biological activity of eachantibody were shown to be almost equal to or greater than those of H0L0.

TABLE 16 Sample ka (1/Ms) kd (1/s) KD (M) H0L0 2.1E+05 8.8E−04 4.2E−09H28L17 6.4E+05 3.3E−04 5.2E−10 H30L17 6.8E+05 5.7E−04 8.3E−10 H34L173.4E+05 1.2E−03 3.6E−09 H42L17 5.7E+05 3.7E−04 6.5E−10 H44L17 6.1E+057.2E−04 1.2E−09 H46L17 2.9E+05 1.3E−03 4.6E−09 H57L17 7.1E+05 5.5E−047.7E−10 H71L17 3.7E+05 1.2E−03 3.3E−09 H78L17 6.1E+05 7.0E−04 1.1E−09H92L17 3.1E+05 1.3E−03 4.1E−09 H97L50 3.6E+05 1.3E−03 3.5E−09 H98L502.9E+05 1.3E−03 4.6E−09

[Example 13] Analysis of the Binding Domain of Anti-NR10 NeutralizingAntibody (1) Preparation of Human/Mouse Wild-Type and Chimeric Antigens

The genes encoding human and mouse wild-type extracellular domains andchimeric extracellular domains of NR10 (hhh (SEQ ID NO: 258), mmm (SEQID NO: 259), hhm (SEQ ID NO: 260), mmh (SEQ ID NO: 261), hmm (SEQ ID NO:262), mhm (SEQ ID NO: 263), and mhh (SEQ ID NO: 264)), were fused to Histag and Myc tag (HHHHHHEQKLISEEDUSEQ ID NO: 287) at their C termini,inserted into an animal expression vector, and transiently expressedusing FreeStyle 293 Expression System (Invitrogen™). Schematic diagramsfor the human/mouse wild-type and chimeric NR10-ECDs are shown in FIG.26.

The human/mouse wild-type and chimeric antigens (hhh, mmm, hhm, mmh,hmm, mhm, and mhh) were purified from culture supernatants by Ni-NTASuperflow column chromatography. Specifically, 1 ml of Ni-NTA Superflow(QIAGEN) was loaded onto Poly-Prep Empty Column (BioRad), and 30 ml ofeach culture supernatant was added thereto. After washing with D-PBS(Dulbecco's phosphate-buffered saline) containing 150 mM sodium chlorideand 20 mM imidazole, the column was eluted with D-PBS containing 150 mMsodium chloride and 250 mM imidazole. The eluted fractions werebuffer-exchanged with D-PBS and concentrated using Amicon-Ultra(Millipore) with a molecular weight cut-off of 10K.

(2) Detection of Binding Antigen by Western Blotting

Each of the prepared human/mouse wild-type and chimeric antigens waselectrophoresed at 0.5 μg/lane on three 4-20% polyacrylamide gels(Daiichi Pure Chemicals Co.). The proteins were electro-transferred ontoPVDF membranes (Millipore) in a semi-dry blotting apparatus, and themembranes were blocked with TBS containing 5% skim milk. One membranewas incubated with 5 μg/ml of H44M58L17 (detection system for humanizedanti-human NR10 antibody); another with 5 μg/ml of ND41 (detectionsystem for mouse anti-human NR10 antibody); and the other one withanti-Myc antibody (SantaCruz, Cat.#sc-789) 500-times diluted with TBScontaining 5% skim milk (detection system for Myc tag) at roomtemperature for one hour.

After washing three times with TBS containing 0.05% Tween™ 20, thesecondary antibodies were incubated with the membranes. Alkalinephosphatase-labeled goat anti-human IgGγ (BIOSOURCE, Cat. #AHI0305) wasused to detect humanized anti-human NR10 antibody; alkalinephosphatase-labeled goat anti-mouse IgG (SantaCruz, Cat. #sc-2008) wasused to detect mouse anti-human NR10 antibody; and alkalinephosphatase-labeled goat anti-rabbit IgG (SantaCruz, Cat. #sc-2057) wasused to detect Myc tag. The reaction was carried out at room temperaturefor one hour. After washing four times with TBS containing 0.05% Tween™20 for three minutes, color development was carried out using BCIP/NBTPhosphatase substrate, 1-Component System (KPL). TBS (Tris-bufferedsaline) used here was prepared by dissolving a pack of TBS (Trisbuffered saline) powder (TaKaRa) in 1 L of distilled water. The resultis shown in FIG. 27.

When the humanized antibody or mouse antibody was used, the binding wasdetected only for hhh, hhm, and hmm, which are NR10 extracellulardomains.

[Referential Example 1] Isolation of Cynomolgus Monkey NR10, OSMR, andIL-31 Genes

Since the cross-reactivity and neutralizing activity in cynomolgusmonkeys were considered important for safety assessment at apre-clinical stage, the cynomolgus monkey NR10 and OSMR genes wereisolated. Primers were designed based on published information of Rhesusmonkey genome and others, and the NR10 and OSMR genes were successfullyamplified by PCR from cynomolgus monkey pancreatic cDNA. The sequencesof the isolated cynomolgus monkey NR10, OSMR, and IL-31 genes are shownin SEQ ID NOs: 65, 69, and 67, respectively, and the amino acidsequences of cynomolgus monkey NR10, OSMR, and IL-31 are shown in SEQ IDNOs: 66, 70, and 68, respectively.

[Referential Example 2] Establishment of NR10- and OSMR-Expressing Ba/F3Cell Lines

The full-length human NR10 cDNA (SEQ ID NO: 75) was inserted into theexpression vector pCOS1 (Biochem. Biophys. Res. Commun. 228, p838-45,1996), and the resulting vector was named pCosNR10.3. An oncostatin Mreceptor cDNA (OSMR, GenBank accession No. NM003999) was isolated by PCRfrom a human placental library, and the expression vector pCos1-hOSMRwas constructed in the same manner. 10 μg each of the vectors weresimultaneously introduced into mouse IL-3-dependent pro-B cell-derivedcell line Ba/F3 by electroporation (BioRad Gene Pulser, 960 μF, 0.33kV). After introduction, human IL-31 (R&D Systems) was added, and thecells were cultured to obtain a cell line (hNR10/hOSMR/BaF3 cell) thatproliferates in an IL-31-dependent manner. Furthermore, the cynomolgusmonkey IL-31 gene (SEQ ID NO: 67) was inserted into a mammalian cellexpression vector and introduced into CHO cell line DG44. The resultingculture supernatant was obtained as cynomolgus monkey IL-31. As withhNR10/hOSMR/BaF3, the full-length cynomolgus monkey NR10 and OSMR geneswere inserted into the expression vector pCOS1 and expressed in Ba/F3cells, and a cynomolgus monkey IL-31-dependent cell line(cynNR10/cynOSMR/BaF3 cell) was established using the culturesupernatant described above.

[Referential Example 3] Establishment of NR10-Expressing CHO Cell Lines

The genes for cytoplasmic domain-lacking human NR10 (SEQ ID NO: 73) andcytoplasmic domain-lacking cynomolgus monkey NR10 (SEQ ID NO: 71) wereeach inserted to a mammalian cell expression vector. The resultingvectors were linearized with a restriction enzyme, and then introducedinto CHO cell line DG44 by electroporation (BioRad Gene Pulser, 25 μF,1.5 kV). After drug selection, NR10-expressing cells were selected andestablished by FCM analysis using anti-human NR10 antibody. The aminoacid sequence encoded by the nucleotide sequence of cytoplasmicdomain-lacking human NR10 gene (SEQ ID NO: 73) is shown in SEQ ID NO:74, and the amino acid sequence encoded by the nucleotide sequence ofcytoplasmic domain-lacking cynomolgus monkey NR10 gene (SEQ ID NO: 71)is shown in SEQ ID NO: 72.

[Referential Example 4] Preparation of NR10 Protein (ExtracellularDomain)

The human NR10 cDNA was used as a template to amplify only theextracellular domain by PCR. The amplified region was then fused to aFLAG tag sequence at the C terminus and inserted to a mammalian cellexpression vector. Ten μg of the linearized vector was introduced intoChinese hamster ovary cell line DG44 by electroporation (BioRad GenePulserII, 25 μF, 1.5 kV). A cell line showing high level expression wasobtained. The supernatant of the cell line cultured on a large scale waspurified using anti-FLAG antibody column (Sigma) and gel filtration toobtain soluble NR10. The nucleotide sequence of soluble NR10 is shown inSEQ ID NO: 77, and the amino acid sequence is shown in SEQ ID NO: 78.

[Referential Example 5] Preparation of Anti-Human NR10 Antibodies

Mice were immunized with human NR10 protein (extracellular domain)(described in Referential Example 4), and hybridomas were prepared by aconventional method. The culture supernatants of these hybridomas wereassessed for the neutralizing activity using the human IL-31-dependentcell line (hNR10/hOSMR/BaF3 cell) described in Referential Example 2,and thereby NA633 which has an NR10-neuralizing activity was obtained.

Furthermore, DNA immunization was carried out by He gas-driven gene gunusing a mammalian expression vector carrying the full-length human NR10gene (SEQ ID NO: 75), and hybridomas were prepared by a conventionalmethod. The culture supernatants of these hybridomas were assessed forthe neutralizing activity using the human IL-31-dependent cell line(hNR10/hOSMR/BaF3 cell) described in Referential Example 2, and therebyND41 which has an NR10-neuralizing activity was obtained.

[Referential Example 6] Preparation of Human Chimeric Antibody

The amino acid sequences of heavy chain and light chain variable regionsof NA633 are shown in SEQ ID NOs: 104 and 108, respectively. The aminoacid sequences of CDR1, CDR2, and CDR3 of the heavy chain variableregion of NA633 are shown in SEQ ID NOs: 105, 106, and 107,respectively, while those of CDR1, CDR2, and CDR3 of the light chainvariable region are shown in SEQ ID NOs: 109, 110, and 111,respectively. Furthermore, a chimeric antibody between these mousevariable regions and human constant region (H chain, γ1; L chain, κ) wasproduced by a conventional method.

[Referential Example 7] Preparation of huPM1-SKSC in which theHeterogeneity of Wild Type IgG2 is Reduced without Loss of Stability

Since the NS22 antibody is an NR10-neutralizing antibody, its binding toFcγ receptor may be unfavorable in consideration of the immunogenicityand adverse effects. A possible method for reducing the binding to Fcγreceptor is to select IgG2 or IgG4 instead of IgG1 as the isotype of theconstant region (Ann Hematol. 1998 June; 76(6): 231-48). From theviewpoint of Fcγ receptor I and retention in plasma, IgG2 has beenconsidered more desirable than IgG4 (Nat Biotechnol. 2007 December;25(12): 1369-72). Meanwhile, when an antibody is developed as apharmaceutical, properties of the protein, particularly homogeneity andstability, are highly important. The IgG2 isotype has been reported tohave very high heterogeneity resulting from the disulfide bonds in thehinge region (J Biol Chem. 2008 Jun. 6; 283(23): 16206-15). It is noteasy and would be more costly to manufacture it as pharmaceutical in alarge scale while maintaining difference in the heterogeneity ofdesired/related substances among products resulting from the above.Accordingly, it is desired that the substance be composed of a singlesubstance as much as possible. Thus, when antibodies of IgG2 isotype aredeveloped as pharmaceuticals, it is preferred to reduce theheterogeneity resulting from disulfide bonds, without lowering thestability.

In order to reduce the heterogeneity of the wild type IgG2, cysteines inthe hinge region and CH1 domain of IgG2 were substituted. As a result ofexamination of various variants, SKSC (SEQ ID NO: 62), which is aconstant region obtained by altering cysteine at position 131 andarginine at position 133 in the EU numbering (Sequences of proteins ofimmunological interest, NIH Publication No. 91-3242) within the H-chainCH1 domain of the wild type IgG2 constant region sequence to serine andlysine, respectively, and altering cysteine at EU-numbering position 219in the H-chain upper hinge to serine could reduce the heterogeneitywithout decreasing the stability. Meanwhile, other possible methods fordecreasing heterogeneity are to alter only cysteine at EU-numberingposition 219 in the H-chain upper hinge to serine, and to alter onlycysteine at EU-numbering position 220 to serine. Thus, constant regionSC (SEQ ID NO: 153) in which cysteine at EU-numbering position 219 inIgG2 has been altered to serine, and constant region CS (SEQ ID NO: 154)in which cysteine at EU-numbering position 220 in IgG2 has been alteredto serine, were produced.

huPM1-SC (SEQ ID NO: 157), huPM1-CS (SEQ ID NO: 158), huPM1-IgG1 (SEQ IDNO: 159), huPM1-IgG2 (SEQ ID NO: 160), and huPM1-SKSC (SEQ ID NO: 161),which were prepared by combining the constant regions produced as above,IgG1 (SEQ ID NO: 60), and IgG2 (SEQ ID NO: 132) with the variable regionof the humanized anti-IL-6 receptor antibody (H chain variable region,huPM1-VH/SEQ ID NO: 155; L chain variable region huPM1-VL/SEQ ID NO:156)(Cancer Res. 1993 Feb. 15; 53(4): 851-6), were used as an H chain,and huPM1-L (SEQ ID NO: 162) was used as an L chain, to produce eachantibody. Each antibody was expressed and purified by the methoddescribed in Example 4.

The antibodies were compared to each other in terms of theheterogeneity. The heterogeneity of huPM1-IgG1, huPM1-IgG2, huPM1-SC,huPM1-CS, and huPM1-SKSC was assessed by cation exchange chromatography.The chromatography was carried out using a ProPac WCX-10 (Dionex)column, 20 mM sodium acetate (pH 5.0) as mobile phase A, and 20 mMsodium acetate/1M NaCl (pH 5.0) as mobile phase B, with an appropriateflow rate and gradient. The result of assessment by cation exchangechromatography is shown in FIG. 12.

As shown in FIG. 12, conversion of the constant region from IgG1 intoIgG2 increased the heterogeneity. In contrast, the heterogeneity wasmarkedly reduced by converting the constant region into SKSC. Whileconstant region SC resulted in considerable reduction of theheterogeneity as in SKSC, constant region CS did not sufficientlyimprove the heterogeneity.

When an antibody is developed as a pharmaceutical, it is generallydesired that the antibody have high stability in addition to lowheterogeneity for the production of stable preparations. Thus, to assessthe stability, the thermal denaturation midpoint temperature (Tm value)was determined by differential scanning calorimetry (DSC) (VP-DSC;Microcal). The thermal denaturation midpoint temperature (Tm value)serves as an indicator of stability. In order to prepare stablepreparations as pharmaceuticals, a higher thermal denaturation midpointtemperature (Tm value) is preferred (J Pharm Sci. 2008 April; 97(4):1414-26). Thus, huPM1-IgG1, huPM1-IgG2, huPM1-SC, huPM1-CS, andhuPM1-SKSC were dialyzed against a solution of 20 mM sodium acetate/150mM NaCl (pH 6.0) (EasySEP; TOMY), and DSC measurement was carried outusing about 0.1 mg/ml of protein at a heating rate of 1° C./min between40 and 100° C. The denaturation curves obtained by DSC are shown in FIG.13. The Tm values of the Fab domains are listed in Table 17 below.

TABLE 17 Name Tm/° C. huPM1-IgG1 94.8 huPM1-IgG2 93.9 huPM1-SC 86.7huPM1-CS 86.4 huPM1-SKSC 93.7

The Tm values of huPM1-IgG1 and huPM1-IgG2 were almost the same, namely,about 94° C. (IgG2 was lower by about 1° C.). Meanwhile, the Tm valuesof huPM1-SC and huPM1-CS were about 86° C., which was significantlylower than those of huPM1-IgG1 and huPM1-IgG2. On the other hand, the Tmvalue of huPM1-SKSC was about 94° C., and almost the same as huPM1-IgG1and huPM1-IgG2. Since the stability of huPM1-SC and huPM1-CS wasmarkedly lower than that of IgG2, huPM1-SKSC in which cysteine in theCH1 domain have also been altered to serine may be more preferred in thedevelopment of pharmaceuticals. The significant decrease in Tm value ofhuPM1-SC and huPM1-CS as compared to IgG2 may be due to thedisulfide-bonding pattern of huPM1-SC and huPM1-CS that is differentfrom that of IgG2.

Furthermore, comparison of the DSC denaturation curves showed that thedenaturation peak for the Fab domain was sharp in huPM1-IgG1 andhuPM1-SKSC, while it was broader in huPM1-SC and huPM1-CS than the abovetwo, and huPM1-IgG2 gave a shoulder peak on the lower temperature sideof the Fab domain denaturation peak. The denaturation peak in DSCgenerally becomes sharp in the case of a single component, but maybecome broad when two or more components with different Tm values(namely, heterogeneity) are present. Thus, it was suggested thathuPM1-IgG2, huPM1-SC, and huPM1-CS contained two or more components, andthe heterogeneity of natural IgG2 was not reduced in huPM1-SC andhuPM1-CS. This finding suggests that cysteines present in both the hingeregion and the CH1 domain are involved in the heterogeneity of naturalIgG2, and it is necessary to alter not only cysteine in the hinge regionbut also that in the CH1 domain to decrease the heterogeneity on DSC.Furthermore, as described above, it is only possible to attain stabilityequivalent to that of natural IgG2 by altering not only cysteine in thehinge region but also that in the CH1 domain.

As described above, as to the constant regions in which theheterogeneity resulting from the hinge region of IgG2 has been reduced,it was discovered that SC and CS, which are constant regions in whichonly cysteine in the hinge region has been substituted with serine, maybe insufficient from the viewpoint of heterogeneity and stability, andthat it is only possible to significantly reduce the heterogeneity whilemaintaining the stability comparable to IgG2 by additionallysubstituting cysteine at EU-numbering position 131 in the CH1 domainwith serine. Such constant regions include SKSC.

[Referential Example 8] Production and Assessment of Optimized, Non-FcγReceptor-Binding Constant Region M14

In the Fcγ receptor-binding domain of IgG2 constant region, the residuesat EU-numbering positions 233, 234, 235, and 236 are of non-bindingtype, while the residues at EU-numbering positions 327, 330, and 331 aredifferent from those of IgG4, which are of non-binding type. Thus, it isnecessary to alter the amino acids at EU-numbering positions 327, 330,and 331 to the sequence of IgG4 (G2Δa in Eur J Immunol. 1999 August;29(8):2613-24). However, since the amino acid at EU-numbering position339 is alanine in IgG4 while it is threonine in IgG2, mere alteration ofthe amino acids at EU-numbering positions 327, 330, and 331 to thesequence of IgG4 will generate a novel non-naturally occurring 9-aminoacid peptide sequence that could be a T-cell epitope peptide, therebycausing a risk of immunogenicity. Thus, it was found that the occurrenceof the novel peptide sequence could be prevented by altering threonineat EU-numbering position 339 in IgG2 to alanine, in addition to thealterations described above. In addition to the mutations describedabove, methionine at EU-numbering position 397 was mutated into valineto improve the stability of IgG2 under acidic condition. Furthermore, inSKSC (SEQ ID NO: 62) produced in Referential Example 7, in which theheterogeneity resulting from the disulfide bonds in the hinge region hasbeen improved, introduction of mutations at positions 131 and 133 willgenerate a novel non-naturally occurring 9-amino acid peptide sequencethat could be a T-cell epitope peptide, thereby causing a risk ofimmunogenicity. Thus, the peptide sequence around positions 131 to 139was converted into the same as IgG1 by mutating glutamic acid atEU-numbering position 137 into glycine and mutating serine atEU-numbering position 138 into glycine. The constant region sequence M14(SEQ ID NO: 129) was produced by introducing all the above mutations.

The expression and purification of huPM1-M14, prepared by usinghuPM1-M14 as an H chain and huPM1-L (SEQ ID NO: 162) as an L chain, wascarried out by the method described in Referential Example 7. Theprepared huPM1-M14 (SEQ ID NO: 163), huPM1-IgG1, and huPM1-IgG2 wereassessed for the heterogeneity using cation exchange chromatography bythe method described in Referential Example 7.

As shown in FIG. 14, the heterogeneity was also reduced in huPM1-M14 asin huPM1-SKSC.

[Referential Example 9] Preparation of huPM1-M58 with Reduced H-ChainC-Terminal Heterogeneity and Improved Pharmacokinetics

Preparation of huPM1-M58 Molecule

huPM1 is an IgG1 antibody. For the heterogeneity in the C-terminalsequence of the H chain of IgG antibody, the deletion of the C-terminallysine residue and the amidation of the C-terminal amino group due todeletion of the two C-terminal amino acids, glycine and lysine, havebeen reported (Anal Biochem. 2007 Jan. 1; 360(1): 75-83). Also in huPM1,while the major component is a sequence in which the C-terminal lysineencoded by the nucleotide sequence has been deleted bypost-translational modification, there are also a minor component inwhich the lysine remains and a minor component in which the C-terminalamino group is amidated due to deletion of both glycine and lysine,which contribute to heterogeneity. Producing a pharmaceutical in a largescale while maintaining the difference in the heterogeneity ofdesired/related substances between products is not easy but ratherresults in increase of cost, and it is thus desired that the substancebe composed of a single substance as much as possible. When an antibodyis developed as a pharmaceutical, reduction of the heterogeneity isdesired. Thus, it is desired that the C-terminal of the H chain has noheterogeneity when developed as pharmaceuticals. It is also desirable toprolong the plasma half-life of the antibody in order to reduce theantibody dose.

Thus, the alterations described below were introduced to prepare a novelconstant region in which the heterogeneity at C-terminal of the H chainhas been reduced, the pharmacokinetics has been improved as compared tohuPM1-IgG1, and the heterogeneity derived from wild-type IgG2 has alsobeen reduced without loss of stability.

Specifically, in huPM1-SKSC, which has high stability and in which theabove-mentioned heterogeneity related to antibodies with IgG2-isotypeconstant regions is reduced, glutamic acid at EU-numbering position 137was substituted with glycine; serine at position 138 with glycine;histidine at position 268 with glutamine; arginine at position 355 withglutamine; and glutamine at position 419 with glutamic acid. In additionto the above substitutions, glycine and lysine at positions 446 and 447were deleted to reduce the heterogeneity of the H-chain C terminus,thereby obtaining huPM1-M58 (SEQ ID NO: 164). huPM1-M58 prepared byusing huPM1-M58 as an H chain and huPM1-L (SEQ ID NO: 162) as an L chainwas expressed and purified by the method described in Example 4.

The huPM1-M58, huPM1-IgG1, and huPM1-IgG2 were assessed for theheterogeneity and stability by the methods described in Example 5 usingcation exchange chromatography and DSC, respectively.

The result of DSC is shown in Table 18. As shown in FIGS. 13 and 16,huPM1-M58 was found to show reduced heterogeneity without loss ofstability as in huPM1-SKSC.

TABLE 18 Name Tm/° C. huPM1-IgG1 94.8 huPM1-IgG2 93.9 huPM1-SKSC 93.7huPM1-M58 93.7Assessment of huPM1-M58 for Plasma Retention

The prolonged retention (slow elimination) of IgG molecule in plasma isdue to the function of FcRn, which is known as a salvage receptor of IgGmolecule (Nat Rev Immunol. 2007 September; 7(9): 715-25). Whenincorporated into endosomes via pinocytosis, IgG molecules bind to FcRnexpressed in endosomes under the acidic conditions within the endosome(approx. pH 6.0). While IgG molecules that are not bound to FcRn aretransferred to and degraded in lysosomes, those bound to FcRn aretranslocated to the cell surface and then released from FcRn into plasmaagain under the neutral conditions in plasma (approx. pH 7.4).

IgG-type antibodies are known to include IgG1, IgG2, IgG3, and IgG4isotypes. The plasma half-lives of these isotypes in human are reportedto be about 36 days for IgG1 and IgG2; about 29 days for IgG3; and 16days for IgG4 (Nat. Biotechnol. 2007 December; 25(12): 1369-72). Thus,the retention of IgG1 and IgG2 in plasma is believed to be the longest.In general, the isotypes of antibodies used as pharmaceutical agents areIgG1, IgG2, and IgG4. Reported methods for further improving thepharmacokinetics of these IgG antibodies include methods for improvingthe above-described binding activity to human FcRn by altering thesequence of IgG constant region (J. Biol. Chem. 2007 Jan. 19; 282(3):1709-17; J. Immunol. 2006 Jan. 1; 176(1): 346-56).

There are species differences between mouse FcRn and human FcRn (Proc.Natl. Acad. Sci. USA. 2006 Dec. 5; 103(49): 18709-14). Therefore, topredict the retention of IgG antibodies having an altered constantregion sequence in human plasma, it may be desirable to assess thebinding to human FcRn and the plasma retention in human FcRn transgenicmice (Int. Immunol. 2006 December; 18(12): 1759-69).

Assessment of the Binding to Human FcRn

FcRn is a complex of FcRn and β2-microglobulin. Oligo-DNA primers wereprepared based on the published human FcRn gene sequence (J. Exp. Med.(1994) 180 (6), 2377-2381). A DNA fragment encoding the whole gene wasprepared by PCR using human cDNA (Human Placenta Marathon-Ready cDNA,Clontech) as a template and the prepared primers. Using the obtained DNAfragment as a template, a DNA fragment encoding the extracellular domaincontaining the signal region (Met1-Leu290) was amplified by PCR, andinserted into an animal cell expression vector (the amino acid sequenceof human FcRn/SEQ ID NO: 165). Likewise, oligo-DNA primers were preparedbased on the published human β2-microglobulin gene sequence (Proc. Natl.Acad. Sci. U.S.A. 99 (26), 16899-16903 (2002)). A DNA fragment encodingthe whole gene was prepared by PCR using human cDNA (Hu-PlacentaMarathon-Ready cDNA, CLONTECH) as a template and the prepared primers.Using the obtained DNA fragment as a template, a DNA fragment encodingthe whole β2-microglobulin containing the signal region (Met1-Met119)was amplified by PCR and inserted into an animal cell expression vector(the amino acid sequence of human β2-microglobulin/SEQ ID NO: 166).

Soluble human FcRn was expressed by the following procedure. Theprepared plasmids for human FcRn and β2-microglobulin were introducedinto the human embryonic kidney cancer-derived cell line HEK293H(Invitrogen) using 10% fetal bovine serum (Invitrogen) by lipofection.The resulting culture supernatant was collected and purified using IgGSepharose 6 Fast Flow (Amersham Biosciences) by the method described inJ. Immunol. 2002 Nov. 1; 169(9):5171-80. Then further purification wascarried out using HiTrap Q HP (GE Healthcare).

The binding to human FcRn was assessed using Biacore 3000. An antibodywas bound to Protein L or rabbit anti-human IgG Kappa chain antibodyimmobilized onto a sensor chip, human FcRn was added as an analyte forinteraction with the antibody, and the affinity (KD) was calculated fromthe amount of bound human FcRn. Specifically, Protein L was immobilizedonto sensor chip CMS (BIACORE) by the amine coupling method using 50 mMNa-phosphate buffer (pH 6.0) containing 150 mM NaCl as the runningbuffer. Then, an antibody was diluted with the running buffer containing0.02% Tween20, and injected and allowed to bind to the chip. Human FcRnwas then injected to assess the binding activity of the antibody to thehuman FcRn.

The affinity was calculated using BIAevaluation software. The obtainedsensorgram was used to calculate the amount of hFcRn bound to theantibody immediately before the end of human FcRn injection. This wasfitted by the steady state affinity method to calculate the affinity ofhuman FcRn for the antibody.

Predictive Assessment of Plasma Retention of huPM1-IgG1 and huPM1-M58 inHuman Using Human FcRn

The binding activities of huPM1-IgG1 and huPM1-M58 to human FcRn wereassessed using BIAcore. As shown in Table 19, the binding activity ofhuPM1-M58 was greater than that of huPM1-IgG1 by about 1.4 times.

TABLE 19 KD (μM) huPM1-IgG1 1.62 huPM1-M58 1.17

Assessment of the Plasma Retention in Human FcRn Transgenic Mice

The pharmacokinetics in human FcRn transgenic mice (B6.mFcRn−/−.hFcRn Tgline 276+/+ mice; Jackson Laboratories) was assessed by the followingprocedure. An antibody was intravenously administered once at a dose of1 mg/kg to mice, and blood was collected at appropriate time points. Thecollected blood was immediately centrifuged at 15,000 rpm for 15 minutesat 4° C. to obtain plasma. The separated plasma was stored in a freezerat −20° C. or below until use. The plasma concentration was determinedby ELISA.

Predictive Assessment of the Plasma Retention of huPM1-IgG1 andhuPM1-M58 in Human Using Human FcRn Transgenic Mice

The plasma retention of huPM1-IgG1 and huPM1-M58 in human FcRntransgenic mice was assessed. As shown in FIG. 17, the resultdemonstrated that the pharmacokinetics of huPM1-M58 was improved ascompared to huPM1-IgG1. It was suggested that the human FcRn-bindingactivity was correlated to the plasma retention in human FcRn transgenicmice.

[Referential Example 10] Measurement of the Affinity in Antigen-AntibodyReaction Using Biacore

Kinetic analysis of the antigen-antibody reaction was carried out usingBiacore T100 (GE Healthcare Biosciences). The antigen-antibodyinteraction was measured by immobilizing rec-Protein A (hereinafterProtein A) (ZYMED) onto a sensor chip, capturing an antibody on theimmobilized Protein A, and then reacting the antigen as an analyte.Various concentrations of rhNR10 were used as the antigen. The kineticparameters, association rate constant k_(a) (1/Ms) and dissociation rateconstant k_(d) (1/s), were calculated from the sensorgrams obtained bythe measurement. Then, K_(D) (M) was determined based on the rateconstants. Each parameter was determined using Biacore T100 EvaluationSoftware version 1.1 (GE Healthcare Biosciences).

Immobilization of Protein A onto Sensor Chip

Protein A was immobilized onto all flow cells of sensor chip CMS (GEHealthcare Bioscinences) by the amine coupling method. The experimentwas carried out using HBS-EP+ (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA,0.05% v/v Surfactant P20) as a running buffer at a flow rate of 10μL/min. The carboxyl groups of carboxymethyl dextran on the sensor chipwere activated with 1004 of a 1:1 mixture of 75 mg/ml EDC(N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 11.5mg/ml NHS (N-hydroxysuccinimide), and Protein A prepared at 50 μg/mlusing 10 mM acetate buffer (pH 4.5) was allowed to flow for reaction.Then, 1004 of 1 M ethanolamine hydrochloride (pH 8.5) was allowed toflow to inactivate the unreacted active groups. Ultimately, about 4000to 5000 RU were immobilized. The experiment was carried out at 25° C. atall times.

Measurement of Affinity in Antigen-Antibody Reaction Between rhNR10 andAntibody Captured on Protein A

The running buffer used was HBS-EP+. Each antibody was prepared at 0.25μg/ml, or prepared so that about 100 RU would bind to Protein A. rhNR10used as an analyte was prepared at 0, 38.5, 77.0, and 154 nM, or at 0,19.25, and 77.01 nM using HBS-EP+. In the measurement, first, theantibody solution was captured on Protein A, and an analyte solution wasreacted at a flow rate of 20 μL/min for three minutes. Then, thesolution was switched to HBS-EP+, and the dissociation phase wasmeasured for five minutes. After measurement of the dissociation phase,the sensor chip was regenerated by washing with 10 mM glycine-HCl (pH1.5). The obtained sensorgrams were kinetically analyzed using theBiacore-specific data analysis software, Biacore T100 EvaluationSoftware Version 1.1.

INDUSTRIAL APPLICABILITY

The anti-NR10 antibodies obtained by the present inventors exhibit aneffective neutralizing activity against NR10, and are useful as, forexample, therapeutic agents for inflammatory diseases.

1. An antibody that recognizes domain 1 of NR10.
 2. The antibody ofclaim 1, which has a neutralizing activity.
 3. The antibody of claim 1or 2, which is a humanized antibody.
 4. An anti-NR10 antibody which isany one of: (1) an antibody comprising a heavy chain variable regionwhich comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 1,CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR3comprising the amino acid sequence of SEQ ID NO: 3; (2) an antibodycomprising the heavy chain variable region of SEQ ID NO: 4; (3) anantibody comprising a light chain variable region which comprises CDR1comprising the amino acid sequence of SEQ ID NO: 5, CDR2 comprising theamino acid sequence of SEQ ID NO: 6, and CDR3 comprising the amino acidsequence of SEQ ID NO: 7; (4) an antibody comprising the light chainvariable region of SEQ ID NO: 8; (5) an antibody comprising the heavychain variable region of (1) and the light chain variable region of (3);(6) an antibody comprising the heavy chain variable region of (2) andthe light chain variable region of (4); (7) an antibody in which one ormore amino acids are substituted, deleted, added, and/or inserted in theantibody of any one of (1) to (6), which has an activity equivalent tothat of the antibody of any one of (1) to (6); and (8) an antibody whichbinds to the same epitope as an epitope bound by the antibody of any oneof (1) to (7).
 5. An anti-NR10 antibody which is any one of: (1) anantibody comprising a heavy chain variable region which comprises CDR1comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising theamino acid sequence of SEQ ID NO: 10, and CDR3 comprising the amino acidsequence of SEQ ID NO: 11; (2) an antibody comprising the heavy chainvariable region of SEQ ID NO: 12; (3) an antibody comprising a lightchain variable region which comprises CDR1 comprising the amino acidsequence of SEQ ID NO: 13, CDR2 comprising the amino acid sequence ofSEQ ID NO: 14, and CDR3 comprising the amino acid sequence of SEQ ID NO:15; (4) an antibody comprising the light chain variable region of SEQ IDNO: 16; (5) an antibody comprising the heavy chain variable region of(1) and the light chain variable region of (3); (6) an antibodycomprising the heavy chain variable region of (2) and the light chainvariable region of (4); (7) an antibody in which one or more amino acidsare substituted, deleted, added, and/or inserted in the antibody of anyone of (1) to (6), which has an activity equivalent to that of theantibody of any one of (1) to (6); and (8) an antibody which binds tothe same epitope as an epitope bound by the antibody of any one of (1)to (7).
 6. An anti-NR10 antibody which is any one of: (1) an antibodycomprising a heavy chain variable region which comprises CDR1 comprisingthe amino acid sequence of SEQ ID NO: 17, CDR2 comprising the amino acidsequence of SEQ ID NO: 18, and CDR3 comprising the amino acid sequenceof SEQ ID NO: 19; (2) an antibody comprising the heavy chain variableregion of SEQ ID NO: 20; (3) an antibody comprising a light chainvariable region which comprises CDR1 comprising the amino acid sequenceof SEQ ID NO: 21, CDR2 comprising the amino acid sequence of SEQ ID NO:22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23; (4) anantibody comprising the light chain variable region of SEQ ID NO: 24;(5) an antibody comprising the heavy chain variable region of (1) andthe light chain variable region of (3); (6) an antibody comprising theheavy chain variable region of (2) and the light chain variable regionof (4); (7) an antibody in which one or more amino acids aresubstituted, deleted, added, and/or inserted in the antibody of any oneof (1) to (6), which has an activity equivalent to that of the antibodyof any one of (1) to (6); and (8) an antibody which binds to the sameepitope as an epitope bound by the antibody of any one of (1) to (7). 7.An anti-NR10 antibody which is any one of: (1) an antibody comprising aheavy chain variable region which comprises CDR1 comprising the aminoacid sequence of SEQ ID NO: 25, CDR2 comprising the amino acid sequenceof SEQ ID NO: 26, and CDR3 comprising the amino acid sequence of SEQ IDNO: 27; (2) an antibody comprising the heavy chain variable region ofSEQ ID NO: 28; (3) an antibody comprising a light chain variable regionwhich comprises CDR1 comprising the amino acid sequence of SEQ ID NO:29, CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and CDR3comprising the amino acid sequence of SEQ ID NO: 31; (4) an antibodycomprising the light chain variable region of SEQ ID NO: 32; (5) anantibody comprising the heavy chain variable region of (1) and the lightchain variable region of (3); (6) an antibody comprising the heavy chainvariable region of (2) and the light chain variable region of (4); (7)an antibody in which one or more amino acids are substituted, deleted,added, and/or inserted in the antibody of any one of (1) to (6), whichhas an activity equivalent to that of the antibody of any one of (1) to(6); and (8) an antibody which binds to the same epitope as an epitopebound by the antibody of any one of (1) to (7).
 8. An antibody orantibody variable region which is any one of: (1) a heavy chain variableregion comprising CDR1 of SEQ ID NO: 196, CDR2 of SEQ ID NO: 197, andCDR3 of SEQ ID NO: 11 (H17); (2) a heavy chain variable regioncomprising CDR1 of SEQ ID NO: 176, CDR2 of SEQ ID NO: 197, and CDR3 ofSEQ ID NO: 11 (H19); (3) a heavy chain variable region comprising CDR1of SEQ ID NO: 196, CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 184(H28, H42); (4) a heavy chain variable region comprising CDR1 of SEQ IDNO: 9, CDR2 of SEQ ID NO: 197, and CDR3 of SEQ ID NO: 184 (H30, H44);(5) a heavy chain variable region comprising CDR1 of SEQ ID NO: 176,CDR2 of SEQ ID NO: 197, CDR3 of SEQ ID NO: 184 (H34, H46); (6) a heavychain variable region comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ IDNO: 198, and CDR3 of SEQ ID NO: 184 (H57, H78); (7) a heavy chainvariable region comprising CDR1 of SEQ ID NO: 176, CDR2 of SEQ ID NO:198, and CDR3 of SEQ ID NO: 184 (H71, H92); (8) a heavy chain variableregion comprising CDR1 of SEQ ID NO: 9, CDR2 of SEQ ID NO: 199, and CDR3of SEQ ID NO: 184 (H97, H98); (9) a light chain variable regioncomprising CDR1 of SEQ ID NO: 200, CDR2 of SEQ ID NO: 170, and CDR3 ofSEQ ID NO: 193 (L11); (10) a light chain variable region comprising CDR1of SEQ ID NO: 201, CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193(L12); (11) a light chain variable region comprising CDR1 of SEQ ID NO:202, CDR2 of SEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L17); (12) alight chain variable region comprising CDR1 of SEQ ID NO: 203, CDR2 ofSEQ ID NO: 170, and CDR3 of SEQ ID NO: 193 (L50); (13) an antibodycomprising the heavy chain variable region of (3) and the light chainvariable region of (11); (14) an antibody comprising the heavy chainvariable region of (4) and the light chain variable region of (11); (15)an antibody comprising the heavy chain variable region of (5) and thelight chain variable region of (11); (16) an antibody comprising theheavy chain variable region of (6) and the light chain variable regionof (11); (17) an antibody comprising the heavy chain variable region of(7) and the light chain variable region of (11); (18) an antibodycomprising the heavy chain variable region of (8) and the light chainvariable region of (12); (19) an antibody in which one or more aminoacids are substituted, deleted, added, and/or inserted in the antibodyof any one of (13) to (18), which has an activity equivalent to that ofthe antibody of any one of (13) to (18); and (20) an antibody whichbinds to the same epitope as an epitope bound by the antibody of any oneof (13) to (18).
 9. An antibody or antibody variable region which is anyone of: (1) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 204 (H17); (2) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 205 (H19); (3) a heavychain variable region comprising the amino acid sequence of SEQ ID NO:206 (H28); (4) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 207 (H30); (5) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 208 (H34), (6) a heavychain variable region comprising the amino acid sequence of SEQ ID NO:209 (H42); (7) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 210 (H44); (8) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 211 (H46); (9) a heavychain variable region comprising the amino acid sequence of SEQ ID NO:212 (H57); (10) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 213 (H71); (11) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 214 (H78); (12) a heavychain variable region comprising the amino acid sequence of SEQ ID NO:215 (H92); (13) a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 216 (H97); (14) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 217 (H98); (15) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:218 (L11); (16) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 219 (L12); (17) a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 220 (L17); (18) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:221 (L50); (19) an antibody comprising the heavy chain variable regionof (3) and the light chain variable region of (17) (H28L17); (20) anantibody comprising the heavy chain variable region of (4) and the lightchain variable region of (17) (H30L17); (21) an antibody comprising theheavy chain variable region of (5) and the light chain variable regionof (17) (H34L17); (22) an antibody comprising the heavy chain variableregion of (6) and the light chain variable region of (17) (H42L17); (23)an antibody comprising the heavy chain variable region of (7) and thelight chain variable region of (17) (H44L17); (24) an antibodycomprising the heavy chain variable region of (8) and the light chainvariable region of (17) (H46L17); (25) an antibody comprising the heavychain variable region of (9) and the light chain variable region of (17)(H57L17); (26) an antibody comprising the heavy chain variable region of(10) and the light chain variable region of (17) (H71L17); (27) anantibody comprising the heavy chain variable region of (11) and thelight chain variable region of (17) (H78L17); (28) an antibodycomprising the heavy chain variable region of (12) and the light chainvariable region of (17) (H92L17); (29) an antibody comprising the heavychain variable region of (13) and the light chain variable region of(18) (H97L50); (30) an antibody comprising the heavy chain variableregion of (14) and the light chain variable region of (18) (H98L50),(31) an antibody in which one or more amino acids are substituted,deleted, added, and/or inserted in the antibody of any one of (19) to(30), which has an activity equivalent to that of the antibody of anyone of (19) to (30); and (32) an antibody which binds to the sameepitope as an epitope bound by the antibody of any one of (19) to (30).10. The anti-NR10 antibody of any one of claims 4 to 9, which is ahumanized antibody.
 11. An antibody, antibody heavy chain, or antibodylight chain, which is any one of: (1) a heavy chain comprising the aminoacid sequence of SEQ ID NO: 222 (H17); (2) a heavy chain comprising theamino acid sequence of SEQ ID NO: 223 (H19); (3) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 224 (H28); (4) a heavychain comprising the amino acid sequence of SEQ ID NO: 225 (H30); (5) aheavy chain comprising the amino acid sequence of SEQ ID NO: 226 (H34);(6) a heavy chain comprising the amino acid sequence of SEQ ID NO: 227(H42); (7) a heavy chain comprising the amino acid sequence of SEQ IDNO: 228 (H44); (8) a heavy chain comprising the amino acid sequence ofSEQ ID NO: 229 (H46); (9) a heavy chain comprising the amino acidsequence of SEQ ID NO: 230 (H57); (10) a heavy chain comprising theamino acid sequence of SEQ ID NO: 231 (H71); (11) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 232 (H78); (12) a heavychain comprising the amino acid sequence of SEQ ID NO: 233 (H92); (13) aheavy chain comprising the amino acid sequence of SEQ ID NO: 234 (H97);(14) a heavy chain comprising the amino acid sequence of SEQ ID NO: 235(H98); (15) a light chain comprising the amino acid sequence of SEQ IDNO: 236 (L11); (16) a light chain comprising the amino acid sequence ofSEQ ID NO: 237 (L12); (17) a light chain comprising the amino acidsequence of SEQ ID NO: 238 (L17); (18) a light chain comprising theamino acid sequence of SEQ ID NO: 239 (L50); (19) an antibody comprisingthe heavy chain of (3) and the light chain of (17) (H28L17); (20) anantibody comprising the heavy chain of (4) and the light chain of (17)(H30L17); (21) an antibody comprising the heavy chain of (5) and thelight chain of (17) (H34L17); (22) an antibody comprising the heavychain of (6) and the light chain of (17) (H42L17); (23) an antibodycomprising the heavy chain of (7) and the light chain of (17) (H44L17);(24) an antibody comprising the heavy chain of (8) and the light chainof (17) (H46L17); (25) an antibody comprising the heavy chain of (9) andthe light chain of (17) (H57L17); (26) an antibody comprising the heavychain of (10) and the light chain of (17) (H71L17); (27) an antibodycomprising the heavy chain of (11) and the light chain of (17) (H78L17);(28) an antibody comprising the heavy chain of (12) and the light chainof (17) (H92L17); (29) an antibody comprising the heavy chain of (13)and the light chain of (18) (H97L50); (30) an antibody comprising theheavy chain of (14) and the light chain of (18) (H98L50); (31) anantibody in which one or more amino acids are substituted, deleted,added, and/or inserted in the antibody of any one of (19) to (30), whichhas an activity equivalent to that of the antibody of any one of (19) to(30); and (32) an antibody which binds to the same epitope as an epitopebound by the antibody of any one of (19) to (30).
 12. A pharmaceuticalcomposition comprising the antibody of any one of claims 1 to
 11. 13.The pharmaceutical composition of claim 12, which is an agent fortreating an inflammatory disease.